Kinase and phosphatase assays

ABSTRACT

Compositions, methods, and kits for detecting and monitoring kinase or phosphatase activity are described. The compositions typically include a peptide, a detectable moiety, and a protease cleavage site. Modification of a peptide by a kinase or phosphatase alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition. Panel assays for determining substrates or modulators of kinase or phosphatase activity are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority under 35 U.S.C.§119(e) to U.S. Provisional Application Ser. No. 60/490,771, filed Jul.29, 2003, which is incorporated by reference in its entirety herein.

TECHNICAL FIELD

This invention relates to kinase and phosphatase assays, and moreparticularly to compositions, methods, and kits useful for monitoringkinase and phosphatase activity.

BACKGROUND

One of the most important classes of intracellular activities ispost-translational modification of proteins. Post-translationalmodification activities modify proteins within living cells to effectchanges in their biological activity and/or function. Major types ofprotein post-translational modification include protein phosphorylation,methylation, prenylation, glycosylation, ubiquitination, sulfation, andproteolysis.

Protein modification by kinases and phosphatases is generally recognizedas an important mechanism for regulating protein function. Proteinkinases modify proteins by the addition of phosphate groups(phosphorylation), primarily on the amino acids tyrosine, serine, orthreonine. Protein phosphatases, in contrast, act to remove thesephosphate groups. Changes in the phosphorylation state of proteins canaffect enzymatic activity, protein localization, and protein-proteininteractions. Such changes can subsequently modulate cellularmetabolism, regulation, growth, and differentiation.

Researchers have found more than 400 human diseases and disordersarising from genetic defects in protein kinases. Thus, the over 600kinases and phosphatases encoded by the human genome representpotentially powerful targets for drugs. Current methods of measuringprotein kinase activity, however, have many disadvantages, hampering theability to rapidly screen kinases for drugs. For example, many currentmethods for measuring kinase activity rely on the incorporation andmeasurement of ³²P into the protein substrates of interest. In wholecells, this necessitates the use of high levels of radioactivity toefficiently label the cellular ATP pool and to ensure that the targetprotein is efficiently labeled with radioactivity. After incubation withone or more test drugs, the cells must be lysed and the protein ofinterest purified to determine its relative degree of phosphorylation.This method requires large numbers of cells, long preincubation times,and careful manipulation and washing steps to avoid artifactualphosphorylation or dephosphorylation. Alternative kinase assay methods,such as those based on phosphorylation-specific antibodies usingELISA-type approaches, involve the difficulty of producing antibodiesthat distinguish between phosphorylated and non-phosphorylated proteins.

There is thus a need for assays to monitor kinase and phosphataseenzymatic activities that are sensitive, simple to use, and adaptable tohigh-throughput screening methods.

SUMMARY

The invention is based on compositions of matter comprising a peptidehaving a motif, such as a recognition motif for a post-translationalmodification activity, and a detectable moiety. The compositions aresurprisingly useful as sensors of post-translational modificationactivities, including kinase and phosphatase activities. Thecompositions can also be used to determine modulators of suchactivities. The invention also relates to methods of determiningsubstrates and modulators of post-translational modification activities.

In one aspect, the invention provides compositions of matter. Thecompositions can include a peptide having a length from five to fiftyamino acids. For example, the peptides can have a length from 8 to 50amino acids, a length from 8 to 25 amino acids, or a length from 8 to 15amino acids. Compositions can include a first detectable moiety, wherethe first detectable moiety is associated with the peptide, e.g., eithercovalently (optionally through a linker (L)) or non-covalently. Suitablelinkers include GABA, diaminopentanyl, and aminohexanoyl groups.

In some embodiments, the compositions can include a second detectablemoiety. Accordingly, in some compositions described herein, a firstdetectable moiety and a second detectable probe moiety can form a darkquenching RET pair. In other embodiments, a first detectable moiety anda second detectable moiety can form a FRET pair. In some embodiments, afirst detectable moiety is 7-hydroxycoumarin-3-carboxamide and a seconddetectable moiety is 5-FAM.

In one aspect, a peptide can contain a motif selected from AIYAA (SEQ IDNO: 1); QDYLS (SEQ ID NO: 4); EIYGV (SEQ ID NO: 7); TX₁YVA, where X₁ canbe G, A, or E (SEQ ID NO: 110); EEYIQ (SEQ ID NO: 17); or DYSQV (SEQ IDNO: 20). A motif can be a recognition motif for a tyrosine kinase andcan be selected from EAIYAAP (SEQ ID NO: 2); DQDYLSL (SEQ ID NO: 5);EEEYIQI (SEQ ID NO: 18); EEIYGVI (SEQ ID NO: 8); LTGYVAR (SEQ ID NO:11); ITAYVAT (SEQ ID NO: 12); ITEYVAT (SEQ ID NO: 13); or GDYSQVL (SEQID NO: 21). Peptides having such recognition motifs include thefollowing: EAEAIYAAPGDK; (SEQ ID NO: 3) GDQDYLSLDK; (SEQ ID NO: 6)EEEEYIQIVK; (SEQ ID NO: 19) EEEIYGVIEK; (SEQ ID NO: 9) GVLTGYVARRK; (SEQID NO: 14) DDEITAYVATRK; (SEQ ID NO: 15) TGIITEYVATRK; (SEQ ID NO: 16)and EGDYSQVLEK. (SEQ ID NO: 22)

In some embodiments, when a recognition motif for a tyrosine kinase isEAIYAAP (SEQ ID NO: 2), the tyrosine kinase can be selected from thegroup Ab11, Ab12, BMX, CSF1R, Csk, EPHB4, Fes/Fps, FGFR1, FGFR4, Fgr,FLT3, Fyn, Hck, IGF1R, IRKβ, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B,c-MET, Src, Src N1, Src N2, SYK, TIE2, TRKa, and YES. Alternatively, ifthe recognition motif for a tyrosine kinase is DQDYLSL (SEQ ID NO: 5),the tyrosine kinase can be selected from CaMKII, CDK7/CycH, CK1δ, IKKα,and IKKβ. In another embodiment, if a recognition motif for a tyrosinekinase is EEIYGVI (SEQ ID NO: 8), the tyrosine kinase can be Ab11, Ab12,BMX, CSF1R, Csk, EPHB4, Fes/Fps, FGFR1, Fgr, FLT3, Fyn, Hck, IGF1R,IRKβ, IRTK, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, SrcN1, Src N2, SYK, TIE2, TRKa, or YES. In yet another embodiment, if arecognition motif for a tyrosine kinase is LTGYVAR (SEQ ID NO: 11), thetyrosine kinase can be CSF1R, FLT3, or c-KIT. In an additionalembodiment, if a recognition motif for a tyrosine kinase is EEEYIQI (SEQID NO: 18), the tyrosine kinase can be EGFR, Zap-70, PDGFR, FGFR4, Abl1, or Lyn B.

In another aspect, a peptide can have a motif selected from RR(S/T)L(SEQ ID NO: 145); L(S/T)TT (SEQ ID NO: 146); L(S/T)LD (SEQ ID NO: 147);RXI(S/T)X₂, where X₁ can be V, A, or Q and X₂ can be V or L (SEQ ID NO:148); TS(S/T)L (SEQ ID NO: 149); X₁(S/T)PX₂ where X₁ can be P or I andX₂ can be G. K, or D (SEQ ID NO: 150); (S/T)X₁X₂VA, where X₁ can be A,E, or Q and X₂ can be Y or H (SEQ ID NO: 151); I(S/T)IAN (SEQ ID NO:152); SIA(S/T)I (SEQ ID NO: 153); (S/T)VPPS*P, where S* is aphosphorylated serine (SEQ ID NO: 154); DX₁(S/T)X₂, where X₁ can be A orE and X₂ can be I or Q (SEQ ID NO: 155); and D(S/T)QV (SEQ ID NO: 156).

In another aspect, a peptide can include a motif selected fromRRX₁(S/T)L, where X₁ can be F, W, or Y (SEQ ID NO: 45); LX₁₍S/T)TT,where X₁ can be F, W, or Y (SEQ ID NO: 48); X₁L(S/T)LD, where X₁ can beF, W, or Y (SEQ ID NO: 51); RX₁X₂(S/T)X₃, where X₁ can be V, A, or Q, X₂can be F, W, or Y, and X₃ can be V or L (SEQ ID NO: 54); TX₁S(S/T)L,where X₁ can be F, W, or Y (SEQ ID NO: 61); X₁X₂(S/T)PX₃ where X₁ can beP or I, X₂ can be F, W, or Y, and X₃ can be Q K, or D (SEQ ID NO: 64);X₁(S/T)X₂X₃VA, where X₁ can be F, W, or Y, X₂ can be A, E, or Q, and X₃can be Y or H (SEQ ID NO: 71); IX₁(S/T)IAN, where X₁ can be F, W, or Y(SEQ ID NO: 78); SIAX₁(S/T)I, where X₁ can be F, W, or Y (SEQ ID NO:81); (S/T)VPPS*P, where S* is a phosphorylated serine (SEQ ID NO: 84);DX₁X₂(S/T)X₃, where X₁ can be A or E, X₂ can be F, W, or Y, and X₃ canbe I or Q (SEQ ID NO: 87); and DX, (S/T)QV, where X₁ can be F, W, or Y(SEQ ID NO: 92).

In certain embodiments, a motif can be selected from RRF(S/T)L (SEQ IDNO: 157); LF(S/T)TT (SEQ ID NO: 158); YL(S/T)LD (SEQ ID NO: 159);RX₁F(S/T)X₂, where X₁ can be V, A, or Q and X₂ can be V or L (SEQ ID NO:160); TFS(S/T)L (SEQ ID NO: 161); X₁F(S/T)PX₂ where X₁ can be P or I andX₂ can be G. K, or D (SEQ ID NO: 162); F(S/T)X₁X₂VA, where X₁ can be A,E, or Q and X₂ can be Y or H (SEQ ID NO: 163); IF(S/T)IAN (SEQ ID NO:164); SIAF(S/T)I (SEQ ID NO: 165); DX₁F(S/T)X₂, where X₁ can be A or Eand X₂ can be I or Q (SEQ ID NO: 166); and DY(S/T)QV (SEQ ID NO: 167).

In another aspect, the invention provides peptides containing motifsthat can be recognition motifs for serine/threonine kinases. Examples ofrecognition motifs for serine/threonine kinase include LRRFSLG (SEQ IDNO: 46); GLFSTTP (SEQ ID NO: 49); DYLSLDK (SEQ ID NO: 52); NRVFSVA (SEQID NO: 55); PRAFSVG (SEQ ID NO: 56); RRQFSLR (SEQ ID NO: 57); RTFSSLA(SEQ ID NO: 62); APFSPGG (SEQ ID NO: 65); HPFSPKK (SEQ ID NO: 66);KIFSPDV (SEQ ID NO: 67); EFTAYVA (SEQ ID NO: 72); IFTEYVA (SEQ ID NO:73); VFTQHVA (SEQ ID NO: 74); RIFSIANS (SEQ ID NO: 79); DSIAFSIV (SEQ IDNO: 82); FSVPPS*PD, where S* is a phosphorylated serine (SEQ ID NO: 85);EDAFSII (SEQ ID NO: 88); EDEFSQN (SEQ ID NO: 89); or EGDYSQV (SEQ ID NO:93). Peptides having such recognition motifs include the following:ALRRFSLGEK (SEQ ID NO: 47); VAPFSPGGRAK (SEQ ID NO: 68); RGGLFSTTPGGTK(SEQ ID NO: 50); KLNRVFSVAC (SEQ ID NO: 58); GDQDYLSLDK (SEQ ID NO: 53);ARPRAFSVGK (SEQ ID NO: 59); RRRQFSLRRKAK (SEQ ID NO: 60); RPRTFSSLAEGK(SEQ ID NO: 63); AKHPFSPKKAK (SEQ ID NO: 69); DDEFTAYVATRK (SEQ ID NO:75);TGIFTEYVATRK (SEQ ID NO: 76); TGVFTQHVATRK (SEQ ID NO: 77);QRIFSIANSIVK (SEQ ID NO: 80); RIDSIAFSIVGK (SEQ ID NO: 83);PRPFSVPPS*PDK, where S* is a phosphorylated Serine (SEQ ID NO: 86);EEDAFSIIGK (SEQ ID NO: 90); REDEFSQNEEK (SEQ ID NO: 91); IIKIFSPDVEK(SEQ ID NO: 70); and EGDYSQVLEK (SEQ ID NO: 22).

When a recognition motif for a serine/threonine kinase is LRRFSLG (SEQID NO: 46), the serine/threonine kinase can be selected from the groupconsisting of Akt1, Akt2, Akt3, Aurora A, CaMKII, CDK2/CycA, CDK3/CycE,CDK7/CycH, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1γ, MSK1, PAK2, PKA, PKG, andROCK. In other embodiments, when a recognition motif for aserine/threonine kinase is GLFSTTP (SEQ ID NO: 49), the serine/threoninekinase can be selected from p38γ, p38δ, and REDK. Alternatively, if arecognition motif for a serine/threonine kinase is NRVFSVA (SEQ ID NO:55), the serine/threonine kinase can be Akt3, AMPK, CaMKII, CDK7/CycH,CHK2, IKKα, MAPKAP-K1α, MAPKAP-K2, MAPKAP-K3, MAPKAP-K5, PAK2, PKA,PKCβII, REDK, ROCK, ROCK2, or SGK1. In another embodiment, if arecognition motif for a serine/threonine kinase is PRAFSVG (SEQ ID NO:56), the serine/threonine kinase can be selected from the groupconsisting of Akt1, Akt2, Akt3, CaMKII, CDK7/CycH, IKK, MAPKAP-K1α/RSK1,MAPKAP-K1β/RSK2, MAPKAP-K1γ/RSK3, MSK1, PAK2, PIM1, PKA, PKG, REDK, andSGK1. A recognition motif for a serine/threonine kinase can be RRQFSLR(SEQ ID NO: 57), where the serine/threonine kinase can be Akt1, Akt2,Akt3, CaMKII, CHK1, CHK2, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1γ, MSK1, p70S6 Kinase, PAK2, PIM1, PKA, PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCζ,PKCη, PKCθ, PKCι, PKG ROCK, ROCK2, or SGK1. In another embodiment, arecognition motif for a serine/threonine kinase is RTFSSLA (SEQ ID NO:62), and the serine/threonine kinase is selected from the groupconsisting of Akt1, CDK2/CycA, CDK6, IKKβ, MAPKAP-K1α, MAPKAP-K1β,MAPKAP-K1γ, p70 S6 Kinase, PAK2, and PKA. A recognition motif for aserine/threonine kinase can be APFSPGG (SEQ ID NO: 65), and theserine/threonine kinase can be selected from the group consisting ofCDK2/CycA, CDK3/CycE, ERK1, ERK2, IKKα, p38β, p38γ, and p38δ.

Any of the compositions described herein can include a protease cleavagesite, such as a chymotrypsin protease cleavage site, a caspase 3protease cleavage site, a cathepsin G protease cleavage site, a trypsinprotease cleavage site, an elastase protease cleavage site, anendoproteinase Asp-N protease cleavage site, or an endoproteinase Glu-Nprotease cleavage site. In certain embodiments, the protease cleavagesite can include a sequence FS, FT, or Y.

The compositions can exhibit a detectable property, such as an opticalproperty, a magnetic property, or a radioactive property. For example,an optical property can be a molar extinction coefficient at anexcitation wavelength, a quantum efficiency, an excitation spectrum, anemission spectrum, an excitation wavelength maximum, an emissionwavelength maximum, a ratio of excitation amplitudes at two wavelengths,a ratio of emission amplitudes at two wavelengths, an excited statelifetime, an anisotropy, a polarization of emitted light, a resonanceenergy transfer, or a quenching of emission at a wavelength. The opticalproperty can be a fluorescent property, e.g., a fluorescence excitationspectrum, a fluorescence emission spectrum, a fluorescence excitationwavelength maximum, a fluorescence emission wavelength maximum, a ratioof fluorescence excitation amplitudes at two wavelengths, a ratio offluorescence emission amplitudes at two wavelengths, a fluorescenceexcited state lifetime, a fluorescence anisotropy, or a quenching offluorescence emission at a wavelength. In certain embodiments, acomposition can exhibit a fluorescence excitation maximum in the rangefrom 250 to 750 nm and a fluorescence emission maximum in the range from450 to 800 nm.

A detectable moiety can be, for example, a fluorescent molecule such as5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide,6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate,dichlorotriazinylaminofluorescein,tetramethylrhodamine-5-isothiocyanate,tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein,5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and7-amino-4-methylcoumarin-3-acetic acid. In other embodiments, adetectable moiety is a binding pair member, e.g., an epitope for anantibody or biotin.

In another aspect, the invention provides a method for characterizing akinase. The method includes the steps of contacting a compositiondescribed previously with a protein kinase under conditions effectivefor the protein kinase to phosphorylate the composition, and measuringthe ability of the protein kinase to phosphorylate the composition.

In yet another aspect, the invention features a method for identifying asubstrate of a kinase. The method includes contacting a compositiondescribed above with a protein kinase; contacting the composition andthe protein kinase with a protease to form a protease mixture; andcomparing a measurable property in the protease mixture with themeasurable property in a control protease mixture lacking the proteinkinase. The composition is identified as a substrate of the proteinkinase if the measurable property in the protease mixture is differentfrom the measurable property in the control protease mixture. ATP can bepresent during the contacting step with the kinase. In some embodiments,a measurable property in the protease mixture is compared with ameasurable property in a control protease mixture lacking ATP, where thecomposition is identified as a substrate of the kinase if the measurableproperty in the protease mixture is different from the measurableproperty in the control protease mixture.

In some embodiments of the method, two or more different compositionsare contacted independently with the protein kinase and ATP during thecontacting step to form two or more kinase mixtures. Each of the kinasemixtures is contacted independently with a protease during thecontacting step with the protease to form two or more protease mixtures.A measurable property in each of the protease mixtures is compared withthe measurable property in a corresponding control mixture. In otherembodiments, two or more different protein kinases are contactedindependently with the composition and the ATP during the contactingstep to form two or more kinase mixtures. Each of the kinase mixtures isthen contacted independently with a protease to form two or moreprotease mixtures, and a measurable property in each of the proteasemixtures is compared with the measurable property in a correspondingcontrol mixture.

The comparison of measurable properties can occur concurrently with theprotease contacting step or after the protease contacting step. Thecontacting step can be completed by inhibiting a proteolytic activity ofthe protease, e.g., by adding a reagent to the protease mixtures or byheating the protease mixtures. The reagent can be aprotinin, PMSF, TPCK,AEBSF, chymotrypsin inhibitor 1, and chymotrypsin inhibitor 2.

The invention also provides a method for identifying a modulator ofactivity of a kinase. In the method, a mixture of a protein kinase, asubstrate for the protein kinase, and a test compound are mixed; themixture is contacted with a protease to form a protease mixture; and ameasurable property in the protease mixture is compared to themeasurable property in a control mixture of the substrate, the proteinkinase, and the protease in the absence of the test compound. The testcompound is identified as a modulator of activity of the kinase if themeasurable property in the protease mixture is different from themeasurable property in the control mixture. ATP can be present duringthe kinase contacting step.

In some embodiments, two or more different test compounds can becontacted independently with the protein kinase, ATP, and the substratein the contacting step to form two or more kinase mixtures. Each of thekinase mixtures is contacted independently with a protease to form twoor more protease mixtures, and a measurable property in each of theprotease mixtures is compared with the measurable property in acorresponding control mixture. In other embodiments, two or moredifferent protein kinases are contacted independently with ATP, the testcompound, and the substrate to form two or more kinase mixtures; each ofthe kinase mixtures is contacted independently with a protease to formtwo or more protease mixtures; and a measurable property in each of theprotease mixtures is compared with the measurable property in acorresponding control mixture. The comparison step can occur during orafter the protease contacting step. The protease contacting step may becompleted as described previously.

In another aspect, the invention provides phosphorylated compositions ofmatter. Such compositions of matter can be useful as substrates forphosphatases. For example, a Y or an S/T in a motif described above maybe phosphorylated, e.g., chemically or enzymatically. In otherembodiments, a Y or an S/T in a recognition motif for a tyrosine kinaseor a S/T kinase, respectively, may be phosphorylated to result in arecognition motif for a protein phosphatase. Examples of a proteinphosphatase recognition motif include LRRFS*LG (SEQ ID NO: 96); GLFS*TTP(SEQ ID NO: 99); DYLS*LDK (SEQ ID NO: 102); NRVFS*VA (SEQ ID NO: 105);PRAFS*VG (SEQ ID NO: 106); RRQFS*LR (SEQ ID NO: 107); RTFSS*LA (SEQ IDNO: 112); APFS*PGG (SEQ ID NO: 115); HPFS*PKK (SEQ ID NO: 116); KIFS*PDV(SEQ ID NO: 117); EFT*AYVA (SEQ ID NO: 122); IFT*EYVA (SEQ ID NO: 123);VFT*QHVA (SEQ ID NO: 124); RIFS*IANS (SEQ ID NO: 129); DSIAFS*IV (SEQ IDNO: 132); FS*VPPS*PD (SEQ ID NO: 135); EDAFS*II (SEQ ID NO: 138);EDEFS*QN (SEQ ID NO: 139), and EGDYS*QV (SEQ ID NO: 143), where S*represents a phosphorylated serine and T* represents a phosphorylatedthreonine.

Examples of peptides comprising phosphatase recognition motifs includeEAEAIY*AAPGDK (SEQ ID NO: 25); GDQDY*LSLDK (SEQ ID NO: 28); EEEEY*IQIVK(SEQ ID NO: 41); EEEIY*GVIEK (SEQ ID NO: 31); GVLTGY*VARRK (SEQ ID NO:36); DDEITAY*VATRK (SEQ ID NO: 37); TGIITEY*VATRK (SEQ ID NO: 38), andEGDY*SQVLEK (SEQ ID NO: 44), where Y* represents a phosphorylatedtyrosine. In other embodiments, a peptide comprising a phosphataserecognition motif has a sequence selected from ALRRFS*LGEK (SEQ ID NO:97); VAPFS*PGGRAK (SEQ ID NO: 118); RGGLFS*TTPGGTK (SEQ ID NO: 100);KLNRVFS*VAC (SEQ ID NO: 108); GDQDYLS*LDK (SEQ ID NO: 103); ARPRAFS*VGK(SEQ ID NO: 109); RRRQFS*LRRKAK (SEQ ID NO: 110); RPRTFSS*LAEGK (SEQ IDNO: 113); AKHPFS*PKKAK (SEQ ID NO: 119); DDEFT*AYVATRK (SEQ ID NO: 125);TGIFT*EYVATRK (SEQ ID NO: 126); TGVFT*QHVATRK (SEQ ID NO: 127);QRIFS*IANSIVK (SEQ ID NO: 130); RIDSIAFS*IVGK (SEQ ID NO: 133);PRPFS*VPPS*PDK (SEQ ID NO: 136); EEDAFS*IIGK (SEQ ID NO: 140);REDEFS*QNEEK (SEQ ID NO: 141); IIKIFS*PDVEK (SEQ ID NO: 120), andEGDYS*QVLEK (SEQ ID NO: 144).

In certain embodiments, a phosphatase recognition motif is EAIY*AAP (SEQID NO:24), and the phosphatase is selected from the group consisting ofPTP1B, LAR, and LCA. Alternatively, a phosphatase recognition motif canbe DQDYLS*L (SEQ ID NO: 27), and the phosphatase can be PP1α, PP2A,PP2B, or PP2C. In other embodiments, a phosphatase recognition motif isLRRFS*LG (SEQ ID NO: 96), and the phosphatase is selected from the groupconsisting of PP1α, PP2A, and PP2C. In yet other embodiments, aphosphatase recognition motif is GLFS*TTP (SEQ ID NO: 99), and thephosphatase is selected from PP1α, PP2A, PP2B, or PP2C. Additionally, aphosphatase recognition motif can be NRVFS*VA (SEQ ID NO: 105), and thephosphatase can be PP1α, PP2A, PP2B, or PP2C; a phosphatase recognitionmotif can be PRAFS*VG (SEQ ID NO: 106), with the phosphatase selectedfrom the group consisting of PP1α, PP2A, and PP2B; the phosphataserecognition motif can be RRQFS*LR, (SEQ ID NO: 107) and the phosphatasecan be PP1α, PP2A, or PP2B; a phosphatase recognition motif can beRTFSS*LA (SEQ ID NO: 112), and the phosphatase can be PP1α, PP2A, orPP2B; a phosphatase recognition motif can be APFS*PGG (SEQ ID NO: 115),and the phosphatase can be PP1α or PP2A; a phosphatase recognition motifcan be EEIY*GVI (SEQ ID NO: 30), and the phosphatase can be PTP1B, LAR,or LCA; or the phosphatase recognition motif can be LTGY*VAR (SEQ ID NO:33), and the phosphatase can be PTP1B, LAR, or LCA.

In an additional aspect, the invention provides a method forcharacterizing a phosphatase. The method includes contacting acomposition described above (e.g., a phosphorylated composition) with aprotein phosphatase under conditions effective for the proteinphosphatase to dephosphorylate the composition, and measuring theability of the protein phosphatase to dephosphorylate the composition.

The invention also provides a method for identifying a substrate of aphosphatase, which includes contacting a composition described abovewith a protein phosphatase; contacting the composition and the proteinphosphatase with a protease to form a protease mixture; and comparing ameasurable property in the protease mixture with a measurable propertyin a control protease mixture lacking phosphatase, where the compositionis identified as a substrate of the phosphatase if the measurableproperty in the protease mixture is different from the measurableproperty in the control protease mixture.

In certain embodiments, two or more different compositions are contactedindependently with the phosphatase to form two or more phosphatasemixture; each of the phosphatase mixtures is contacted independentlywith a protease to form two or more protease mixtures; and a measurableproperty in each of the protease mixtures is compared with themeasurable property in a corresponding control mixture. In otherembodiments, two or more different phosphatases are contactedindependently with the composition; each of the phosphatase mixtures iscontacted independently with a protease to form two or more proteasemixtures; and a measurable property in each of the protease mixtures iscompared with the measurable property in a corresponding controlmixture.

The invention also provides a method for identifying a modulator ofactivity of a phosphatase, including contacting a mixture of a proteinphosphatase, a substrate for the protein phosphatase, and a testcompound to form a phosphatase mixture; contacting the phosphatasemixture with a protease to form a protease mixture; and comparing ameasurable property in the protease mixture to the measurable propertyin a control protease mixture lacking the test compound, where the testcompound is identified as a modulator of activity of the phosphatase ifthe measurable property in the protease mixture is different from themeasurable property in the control mixture. In certain embodiments, twoor more different test compounds may be contacted independently with thephosphatase and the substrate to form two or more phosphatase mixtures;each of the phosphatase mixtures may be contacted independently with aprotease to form two or more protease mixtures; and a measurableproperty in each of the protease mixtures may be compared with themeasurable property in a corresponding control mixture. In otherembodiments, two or more different phosphatases are contactedindependently with the test compound and the substrate to form two ormore phosphatase mixtures; each of the phosphatase mixtures is contactedindependently with a protease to form two or more protease mixtures; anda measurable property in each of the protease mixtures is compared withthe measurable property in a corresponding control mixture.

In an additional aspect, the invention provides articles of manufacture.An article of manufacture can include packaging matter and a compositionof matter described herein associated with the packaging material. Thearticle can further comprise a protein kinase or a protein phosphatase;a protease; ATP; and/or buffers.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic indicating the effect on a fluorescence signal ofdifferential sensitivity of a composition to a protease. In the KinaseReaction, a substrate for a kinase (e.g., a composition of matteraccording to the present invention) is phosphorylated by the kinase. Asis shown, the substrate in both the unphosphorylated and phosphorylatedstates exhibits FRET between the donor and acceptor fluorophores on theN- and C-termini of the substrate. In the Development (Protease)Reaction, the phosphorylated and unphosphorylated substrates are exposedto a protease, which differentially cleaves the unphosphorylatedsubstrate relative to the phosphorylated substrate. As shown in theDetection panel, cleavage of the unphosphorylated substrate disruptsFRET between the donor and acceptor fluorophores, and results in ameasurable change in the ratio of the donor fluorescence emission valuerelative to the acceptor fluorescence emission value.

FIG. 2 is a flow chart for identifying modulators of the activity of aTyrosine (FIG. 2A) or Serine/Threonine (FIG. 2B) kinase.

FIG. 3 illustrates the derivation of kinetic parameters for Abl 1kinase.

FIG. 4 demonstrates the dependence of % phosphorylation on Akt 1 kinaseconcentration.

FIG. 5 demonstrates the dependence of % phosphorylation on Abl 1 kinaseconcentration.

FIG. 6 demonstrates dose-dependent inhibition of Abl 1 kinase.

FIG. 7 demonstrates dose-dependent inhibition of Akt 1 kinase.

FIG. 8 demonstrates % phosphorylation data with varying concentrationsof PKA kinase.

FIG. 9 demonstrates % phosphorylation data with varying concentrationsof PKCα.

DETAILED DESCRIPTION

The present invention is based in part on the finding that compositionsof matter that include peptides and detectable moieties can be designedto act as sensors of post-translational modification activities,including kinase or phosphatase activity. Post-translationalmodification of a composition containing a peptide results in amodulation of the rate and efficiency of cleavage of the modifiedpeptide by a protease as compared to the non-modified peptide. Theattachment of at least one detectable moiety to the peptide in thecomposition couples the cleavage of the peptide in the composition to achange in a detectable property of the composition that can be used tomonitor post-translational activity in a sample and to assay formodulators of a post-translational activity. See FIG. 1.

Compositions of the present invention can be used in assay methods,particularly methods for high-throughput and miniaturized screeningsystems for drug discovery and profiling. In addition, methods and kitsdescribed herein typically exhibit a large dynamic range, high Z′-factorvalues, and increased sensitivity by employing a ratiometric analysis toeliminate well-to-well variations. Finally, methods of the presentinvention can be performed under near initial velocity conditions andproduce accurate IC₅₀ data for kinase and phosphatase inhibitors.

Definitions

Generally, the nomenclature used herein and many of the fluorescence,computer, detection, chemistry, and laboratory procedures describedherein are commonly employed in the art.

Abbreviations: t-Boc, tert-butyloxycarbonyl; Bzl, benzyl; CaMK,calmodulin dependent kinase; CKI, casein kinase 1; PDGF, plateletderived growth factor; Fmoc, fluorenylmethyloxycarbonyl; EGF, epidermalgrowth factor; ELISA, enzyme-linked immuno absorbant assay; FGF,fibroblast growth factor; HF, hydrogen fluoride; HOBT,N-Hydroxybenzotriazole; PyBop,Benzotriazole-I-yl-oxy-tris-pyyrolidino-phosphonium hexafluorophosphate;TFA, trifluoroacteic acid; FITC, fluorescein isothiocyanate; RET,resonance energy transfer; FRET, fluorescence resonance energy transfer;FAM, carboxyfluorescein.

As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings:

The term “RET” means resonance energy transfer, and refers to theradiationless transmission of an energy quantum from its site ofabsorption to the site of its utilization in a molecule, or system ofmolecules, by resonance interaction between chromophores, over distancesconsiderably greater than interatomic, without substantial conversion tothermal energy, and without the donor and acceptor coming into kineticcollision. A donor is a moiety that initially absorbs energy (e.g.,optical energy), and an acceptor is the moiety to which the energy issubsequently transferred. Fluorescence resonance energy transfer (FRET)and time-resolved fluorescence resonance energy transfer (TR-FRET) aretypes of RET.

The term “acceptor” refers to a chemical or biological moiety thatoperates via resonance energy transfer, e.g., a quencher. In RETapplications, acceptors may re-emit energy transferred from a donorfluorescent moiety as fluorescence (e.g., FRET) and are “acceptorfluorescent moieties.” As used herein, such a donor fluorescent moietyand an acceptor fluorescent moiety are referred to as a “FRET pair.”Examples of acceptors include coumarins and related fluorophores;xanthenes such as fluoresceins; fluorescent proteins; rhodols, andrhodamines; resorufins; cyanines; difluoroboradiazaindacenes; andphthalocyanines. In other RET applications, acceptors generally do notre-emit the transferred energy and are sometimes referred to as “darkquenchers.” A fluorescent donor moiety and a dark quenching acceptormoiety may be referred to herein as a “dark quenching RET pair.”Examples of dark quenchers include indigos; benzoquinones;anthraquinones; azo compounds; nitro compounds; indoanilines; and di-and triphenylmethanes.

The term “quencher” refers to a molecule or part of a compound that iscapable of reducing light emission (e.g. fluorescence emission) from adetectable moiety. Such reduction includes reducing the emission oflight after the time when a photon is normally emitted from afluorescent moiety. Quenching may occur by any of several mechanisms,including resonance energy transfer (RET), fluorescence resonance energytransfer (FRET), photo-induced electron transfer, paramagneticenhancement of intersystem crossing, Dexter exchange coupling, darkquenching, and excitation coupling (e.g., the formation of darkcomplexes). Preferred quenchers include those that operate by RET,particularly FRET.

The term “bead” refers to a substantially spherical particle such as asphere or microsphere. Beads may be used within a wide size range.Preferred beads are typically within the range of 0.01 to 100 μm indiameter. Beads may be composed of any material and may comprisefluorescent, luminescent, electro-luminescent, chemo-luminescent,magnetic, or paramagnetic probes. Such beads are commercially availablefrom a variety of sources including Molecular Probes, Sigma, andPolysciences.

The terms “cleavage site,” “protease cleavage site,” and “protease site”are used interchangeably and refer to an amide bond that can be cleavedby a protease and one or more amino acids on either side of the bond.The designations “P₁”, “P₂”, “P₃” etc. refer to the amino acid positions1 amino acid, 2 amino acids and 3 amino acids N-terminal to the bond.The designations “P′₁”, “P′₂”, “P′₃” etc. refer to the amino acidspositions 1 amino acid, 2 amino acids and 3 amino acids C-terminal tothe bond, as shown below:

The term “detectable moiety” refers to a chemical moiety useful as amarker, indicator, or contrast agent. A detectable moiety may be capableof being detected by absorption spectroscopy, luminescence spectroscopy,fluorescence spectroscopy, magnetic resonance spectroscopy (e.g., MRI),or radioisotope detection. The term “fluorescent moiety” refers to adetectable moiety that can absorb electromagnetic energy and is capableof at least partially re-emitting some fraction of that energy aselectromagnetic radiation. Suitable fluorescent moieties include, butare not limited to, coumarins and related dyes, xanthene dyes such asfluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, semiconductor fluorescent nanocrystals,fluorescent proteins, and fluorescent europium and terbium complexes andrelated compounds. In some embodiments, a detectable moiety can be amember of a specific binding pair, or can be associated (e.g.,covalently) with a member of a specific binding pair. Specific bindingpairs are pairs of molecules that are capable of specific interactionwith one another, e.g., have an affinity for one another. For example, aspecific binding pair can be ligand-protein binding pairs, e.g.,enzyme-substrate, biotin-streptavidin, or epitope-antibody bindingpairs. A binding pair that includes a detectable moiety has a largerapparent size than a corresponding binding pair that does not include adetectable moiety, and a larger apparent size than either member of thebinding pair alone. Complexes of binding pairs can be detected by amethod described herein or by other methods known to those of skill inthe art, e.g., in an immunoassay format, a gel shift assay, or achromatographic assay.

The term “motif” refers to an amino acid sequence at least five aminoacids in length. In some embodiments, a motif can be a “recognitionmotif” for a phosphatase or a kinase, i.e., an amino acid sequence thatis effective as a substrate for a protein phosphatase or protein kinase.In some embodiments, a recognition motif may be modified from anaturally existing sequence by at least one amino acid substitution. Insome embodiments, the affinity (apparent K_(d)) of a kinase orphosphatase for a recognition motif is about 1 mM or less, or about 10μM or less, or about 1 μM or less, or about 0.1 μM or less. Arecognition motif need not be an optimal or preferred recognition motif,but encompasses sequences whose phosphorylation by a kinase can bedetected or whose de-phosphorylation by a phosphatase can be detected.In some embodiments, a recognition motif overlaps with or encompasses aprotease cleavage site. In other embodiments, a protease cleavage sitedoes not overlap or encompass a recognition motif.

The term “modulates” refers to partial or complete enhancement orinhibition of an activity or process (e.g., by attenuation of rate orefficiency).

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, peptide, hormone, polysaccharide, lipid), anorganic molecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian, includinghuman) cells or tissues. Modulators may be evaluated for potentialactivity as inhibitors or enhancers (directly or indirectly) of abiological process or processes (e.g., agonist, partial antagonist,partial agonist, inverse agonist, antagonist, antineoplastic agents,cytotoxic agents, inhibitors of neoplastic transformation or cellproliferation, cell proliferation-promoting agents, and the like) byinclusion in screening assays described herein. The activity of amodulator may be known, unknown, or partially known.

The term “non-naturally occurring” refers to the fact that an object,compound, or chemical cannot be found in nature. For example, a peptideor polynucleotide that is present in an organism (including viruses)that can be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory is naturally-occurring,while such a peptide or polynucleotide that has been intentionallymodified by man is non-naturally occurring.

The term “optical property” refers to a property of a composition,compound, or moiety and can be any one of the following: a molarextinction coefficient at an appropriate excitation wavelength, afluorescent or luminescent quantum efficiency, a shape of an excitationspectrum or emission spectrum, an excitation wavelength maximum oremission wavelength maximum, a ratio of excitation amplitudes at twodifferent wavelengths, a ratio of emission amplitudes at two differentwavelengths, an excited state lifetime, a fluorescent anisotropy, or anyother measurable optical property derived from the composition,compound, or moiety, either spontaneously or in response toelectromagnetic, electrical, or chemical stimulation or reaction. Onetype of optical property is a fluorescent property, which refers to anoptical property that can be detected using fluorescence-basedtechniques. The fluorescent property can be any one of the following: amolar extinction coefficient at an appropriate excitation wavelength, afluorescent quantum efficiency, a shape of an excitation or emissionspectrum, an excitation wavelength maximum, an emission magnitude at anywavelength during or at one or more times after excitation of afluorescent moiety, a ratio of excitation amplitudes at two differentwavelengths, a ratio of emission amplitudes at two differentwavelengths, an excited state lifetime, a fluorescent anisotropy, or anyother measurable property of a fluorescent moiety. In some embodiments,a fluorescent property refers to fluorescence emission or thefluorescence emission ratio at two or more wavelengths.

The term “peptide” refers to a polymer of two or more amino acids joinedtogether through amide bonds. Amino acids may be natural or unnaturalamino acids, including, for example, beta-alanine, phenylglycine, andhomoarginine. For a review, see Spatola, A. F., in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983). All of the amino acids used inthe present invention may be either the D- or L-isomer. Chemicallymodified or substituted amino acids including phosphorylated (e.g.,phospho-serine (phosphorylated at the hydroxyl of the side chain),phospho-tyrosine (phosphorylated at the OH of the side-chain phenylring), and phospho-threonine (phosphorylated at the hydroxyl of the sizechain)), sulfated, methylated, or prenylated amino acids can also beused to create peptides for specific applications.

The terms “post-translational modification” and “post-translational typemodification” are used interchangeably and refer to enzymatic ornon-enzymatic modification of one or more amino acid residues in apeptide. Typical modifications include phosphorylation,dephosphorylation, glycosylation, methylation, sulfation,ubiquitination, prenylation, and ADP-ribsoylation. Preferredpost-translational type modifications include phosphorylation anddephosphorylation. The term post-translational modification includesnon-covalent type modifications that may affect protein activity,structure, or function, such as protein-protein interactions or thebinding of allosteric modulators, other modulators, or second messengerssuch as calcium, cAMP, or inositol phosphates to the motif, recognitionmotif, or peptide.

The term “test compound” refers to a compound to be tested by one ormore screening method(s) of the invention, e.g., to determine if it is aputative modulator of a kinase or phosphatase. A test compound can beany chemical, such as an inorganic chemical, an organic chemical, aprotein, a peptide, a carbohydrate, a lipid, or a combination thereof.Typically, various predetermined concentrations (e.g., variousdilutions) of test compounds are used for screening, such as 0.01micromolar, 1 micromolar, and 10 micromolar. Test compound controls caninclude the measurement of a signal in the absence of the test compoundor a comparison to a compound known to modulate the target activity.

Compositions

Compositions of the present invention include a peptide. Peptides of theinvention can have a length from five to fifty amino acids and caninclude. one or more motifs. Typically, a motif is five amino acids orlonger in length. A motif can be a recognition motif, e.g., for atyrosine kinase, a serine/threonine kinase, or a phosphatase.Compositions of the present invention can include a first detectablemoiety, and in some embodiments, a second detectable moiety.Compositions of the present invention can include a protease cleavagesite.

Kinases and Phosphatases

In general, protein kinases act on peptides by adding a phosphate groupto a free hydroxyl group on an amino acid (a process known asphosphorylation), primarily on the amino acids tyrosine, serine, orthreonine. The protein kinases that enzymatically catalyze thesereactions may be classified into a number of distinct families based onstructural and functional properties. Kinases within a family may have asimilar overall topology, similar modes of regulation, and/or similarsubstrate specificities (e.g., see Table 1 of U.S. Pat. No. 6,410,255).For example, members of the AGC (protein kinase A, G or C) families ofkinases may prefer phosphorylation recognition motifs with basic aminoacids (e.g., R or K), while those in the CMGC group may prefer prolinecontaining motifs.

Another family of kinases are the Serine/Threonine kinases, whichphosphorylate serine or threonine amino acids, and Tyrosine kinases,which phosphorylate tyrosine amino acids.

Serine/Threonine (S/T) kinases suitable for use in the present inventioninclude, without limitation, the following: Akt1, Akt2, Akt3, Aurora A,BARKIGRK2, CaMKII, CaMKIIa, CDK1/Cyc B, CDK2/CycA, CDK4/CAK, CDK3/CycE,CDK6/CAK, CDK7/CycH, CK1δ, CKIIα, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1γ,MSK1, PAK2, PKA, PKG, ROCK, ROCK2, ERK1, ERK2, ERK5, GSK-3α, MLCK, mTOR,NEK2, IKKα, IKK43, p38β, p38γ, p38δ, REDK, AMPK, MAPKAP-K2, MAPKAP-K3,MAPKAP-K5, SGK1, PIM1, CHK1, CHK2, PKCα, PKCβI, PKCβII, PKCγ, PKCδ,PKCε, PKCζ, PKCη, PKCθ, PKCι, Raf-1, and p70 S6 Kinase.

Tyrosine kinases suitable for use in the present invention include,without limitation, the following: Abl1, Abl2, BMX, Brk, CSF1R, Csk,Erb-B2, EGFR, EphB4, Fes/Fps, FGFR1, FGFR3, FGFR4, Fgr, FLT3, Fyn, FynT,HCK, Hyl, IGF1R, IRKβ, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET,Src, Src N1, Src N2, SYK, Tec, TIE2, TRKA, VEGFR-1/Flt, YES, and IRTK.Tyrosine kinases characterized as receptor tyrosine kinases, and thatare also suitable, include EGFR, EphB4, Erb-B2, FGFR1, FGFR3, FGFR4,FLT3/FLT2, FMS/CSFR1, IGF1R, KDR, c-KIT, c-MET, TIE2, TRKA, andVEGFR-1/Flt. Tyrosine protein kinases characterized as soluble tyrosineprotein kinases are also suitable, and include Abl1, Abl2, Brk, BMX,Csk, Fes/Fps, Fgr, Fyn, FynT, Hck, Hyl, ITK, Jak3, Lck, LynA, LynB, Src,Src, N1, SYK, Tec, and YES. CLK1 is a dual protein kinase and may alsobe used in the present invention.

Eukaryotic protein phosphatases are structurally and functionallydiverse enzymes that have been classified into three distinct genefamilies. Two of these families dephosphorylate phosphoserine andphosphothreonine amino acids, whereas the protein tyrosine phosphatasefamily (PTPs) dephosphorylates phosphotyrosine amino acids. A subfamilyof the PTPs, the dual specificity phosphatases, dephosphorylates allthree phosphoamino acids. Within each family, catalytic domains arereported to be highly conserved, with functional diversity endowed byregulatory domains and subunits.

The protein serine or threonine phosphatases type 1 and 2A reportedlyaccount for as much as 95% of the phosphatase activity in cell extracts(Brautigan and Shriner, Methods. Enzymol. 159: 339-346 (1988)). Theseenzymes have broad substrate specificities and may be regulated in vivothrough targeting of the enzymes to discrete sub-cellular localizations.The total number of protein tyrosine phosphatases encoded in themammalian genome has been estimated at between 500 and approximately2000.

Phosphatases for use in the present invention include, withoutlimitation, the following: PTEN, PTP-meg 1, T-cell-PTP N2, PTP 1B, LAR,LCA, PP1α, PP2A, PP2B, and PP2C.

Motifs and Peptides for Measuring Protein Phosphorylation andDephosphorylation

Motifs suitable for detecting or measuring kinase or phosphataseactivity generally include an amino acid residue which, when modified,modulates the rate of cleavage of a composition by a protease ascompared to the unmodified composition. Typically, peptides of theinvention include a motif having a single protease cleavage site(although more may be useful in some applications) and are soluble (e.g.0.1 mg/ml or greater) in aqueous solution. As one of skill in the artwill recognize, the design and size of peptides for specificcompositions and the choice of a particular protease is dependent uponthe application for which the composition is to be used. For example,for resonance energy transfer type applications, a peptide willtypically be in the range of 5 to 50 amino acids in length, or 8 to 50amino acids in length, or 8 to 25 amino acids in length, or 8 to 15amino acids in length. For polarization-based applications, these andlarger large peptides (e.g., for example 50 to 100 amino acids inlength, and up to and including entire protein domains) may bedesirable.

Peptides suitable for the invention may include basic amino acids,particularly at the termini, to enhance solubility. In addition, in someembodiments, a peptide can include a C-terminal K in order to provide alocus for conjugation to a detectable moiety (e.g., a fluoresceinderivative). A protease cleavage site can be located at any position ina peptide, including within a motif or recognition motif. A motif,recognition motif, or protease cleavage site may be located at anyposition within a peptide with respect to a first or second detectablemoiety.

Tyrosine Phosphorylation or Dephosphorylation

Compositions for detecting and monitoring tyrosine kinase activityincorporate a motif (e.g., a recognition motif for a tyrosine kinase)into a peptide, and typically have a single Tyr (Y) as the only aromaticamino acid in the composition. It may also be preferable in certaincases to eliminate or reduce the number of negatively charged aminoacids in the P′₁, P′₂ or P′₃ positions. Phosphorylation of a tyrosineamino acid by a tyrosine-directed protein kinase activity modulates therate of hydrolysis of the composition by a protease (e.g., chymotrypsin)as compared to the non-phosphorylated composition. Illustrative examplesof recognition motifs and peptide substrates for tyrosine kinases areshown in Table 2 of U.S. Pat. No. 6,410,255 for use with the proteasechymotrypsin. Other illustrative motifs, recognition motifs, andpeptides for tyrosine kinases are shown in Table 1, below. TABLE 1Illustrative Illustrative Recognition Peptide Motif Motif Sequence AIYAAEAIYAAP EAEAIYAAPGDK (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) QDYLSDQDYLSL GDQDYLSLDK (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) EIYGVEEIYGVI EEEIYGVIEK (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 9) TX₁YVA,where X₁ can LTGYVAR; GVLTGYVARRK; be G, A, or E (SEQ ID NO: 11) (SEQ IDNO: 14) (SEQ ID NO: 10) ITAYVAT; DDEITAYVATRK; (SEQ ID NO: 12) (SEQ IDNO: 15) ITEYVAT TGIITEYVATRK (SEQ ID NO: 13) (SEQ ID NO: 16) EEYIQEEEYIQI EEEEYIQIVK (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) DYSQVGDYSQVL EGDYSQVLEK (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22)

Compositions for detecting protein tyrosine phosphatase activityincorporate a motif (e.g., a recognition motif for a tyrosine kinase)into a peptide, where one or more tyrosine amino acids in the motif arephosphorylated. Dephosphorylation of a tyrosine amino acid in suchcompositions by a tyrosine-directed protein phosphatase activitymodulates the rate of hydrolysis by a protease (e.g., chymotrypsin) ascompared to the phosphorylated composition. Illustrative phosphatasemotifs, recognition motifs, and peptides are shown in Table 2, below,where Y* indicates a phosphorylated tyrosine. TABLE 2 IllustrativeIllustrative Recognition Peptide Motif Motif Sequence AIY*AA EAIY*AAPEAEAIY*AAPGDK (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) QDY*LSDQDY*LSL GDQDY*LSLDK (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28)EIY*GV EEIY*GVI EEEIY*GVIEK (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID NO:31) TX₁Y*VA, where X₁ LTGY*VAR; GVLTGY*VARRK; can be G, A, or E (SEQ IDNO: 33) (SEQ ID NO: 36) (SEQ ID NO: 32) ITAY*VAT; DDEITAY*VATRK; (SEQ IDNO: 34) (SEQ ID NO: 37) ITEY*VAT TGIITEY*VATRK (SEQ ID NO: 35) (SEQ IDNO: 38) EEY*IQ EEEY*IQI EEEEY*IQIVK (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQID NO: 41) DY*SQV GDY*SQVL EGDY*SQVLEK (SEQ ID NO: 42) (SEQ ID NO: 43)(SEQ ID NO: 44)Serine/Threonine (SIT) Phosphorylation or Dephosphorylation

Compositions for measuring serine or threonine kinase activitiesincorporate a motif (e.g., a recognition motif for a S/T kinase)typically containing a single aromatic amino acid (Tyr, Trp or Phe)generally within about three amino acids of a serine or threonine aminoacid. A serine or threonine amino acid is phosphorylated by anappropriate serine or threonine specific kinase. It may be preferable incertain cases (depending on the protease selected) to eliminate orreduce the number of negatively charged amino acids (e.g. Asp or Gluamino acids) in the P′₁, P′₂ or P′₃ positions to ensure that serine orthreonine phosphorylation provides a large modulation in proteolyticsensitivity of the composition upon phosphorylation. Examples ofillustrative recognition motifs and peptides are provided in Table 3 ofU.S. 6,410,255 for use with chymotrypsin. Illustrative motifs,recognition motifs, and peptides for S/T kinases are also shown in Table3, below. TABLE 3 Illustrative Illustrative Recognition Peptide MotifMotif Sequence RRX₁(S/T)L, where X₁ can be F, W, LRRFSLG ALRRFSLGEK or Y(SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47) LX₁(S/T)TT, where X₁ canbe F, W, GLFSTTP RGGLFSTTPGGTK or Y (SEQ ID NO: 48) (SEQ ID NO: 49) (SEQID NO: 50) X₁L(S/T)LD, where X₁ can be F, W, DYLSLDK (SEQ ID NO: 52)GDQDYLSLDK (SEQ ID NO: 53) or Y (SEQ ID NO: 51) RX₁X₂(S/T)X₃, where X₁can be V, NRVFSVA, (SEQ ID NO: 55) KLNRVFSVAC, (SEQ ID NO: 58) A, or Q,X₂ can be F, W, or Y, and PRAFSVG, (SEQ ID NO: 56) ARPRAFSVGK, (SEQ IDNO: 59) X₃ can be V or L (SEQ ID NO: 54) RRQFSLR (SEQ ID NO: 57)RRRQFSLRRKAK (SEQ ID NO: 60) TX₁S(S/T)L, where X₁ can be F, W, RTFSSLA(SEQ ID NO: 62) RPRTFSSLAEGK or Y (SEQ ID NO: 61) (SEQ ID NO: 63)X₁X₂(S/T)PX₃ where X₁ can be P or APFSPGG, (SEQ ID NO: 65) VAPFSPGGRAK,I, X₃ can be F, W, or Y, and X₂ can HPFSPKK, (SEQ ID NO: 66) (SEQ ID NO:68) be G, K, or D (SEQ ID NO: 64) KIFSPDV (SEQ ID NO: 67) AKHPFSPKKAK,(SEQ ID NO: 69) IIKIFSPDVEK, (SEQ ID NO: 70) X₁(S/T)X₂X₃VA, where X₁ canbe F, EFTAYVA, (SEQ ID NO: 72) DDEFTAYVATRK, W, or Y, X₂ can be A, E, orQ, and IFTEYVA, (SEQ ID NO: 73) (SEQ ID NO: 75) X₃ can be Y or H (SEQ IDNO: 71) VFTQHVA (SEQ ID NO: 74) TGIFTEYVATRK, (SEQ ID NO: 76)TGVFTQHVATRK (SEQ ID NO: 77) IX₁(S/T)IAN, where X₁ can be F, W, RIFSIANS(SEQ ID NO: 79) QRIFSIANSIVK or Y (SEQ ID NO: 78) (SEQ ID NO: 80)SIAX₁(S/T)I, where X₁ can be F, W, DSIAFSIV (SEQ ID NO: 82)RIDSIALFSIVGK or Y (SEQ ID NO: 81) (SEQ ID NO: 83) (S/T) VPPS*P, whereS* is a FSVPPS*PD, where S* is a PRPFSVPPS*PDK, where S* isphosphorylated serine phosphorylated serine, a phosphorylated Serine(SEQ ID NO: 84) (SEQ ID NO: 85) (SEQ ID NO: 86) DX₁X₂(S/T)X₃, where X₁can be A EDAFSII, (SEQ ID NO: 88) EEDAFSIIGK, (SEQ ID NO: 90) or E, X₂can be F, W, or Y, and X₃ EDEFSQN (SEQ ID NO: 89) REDEFSQNEEK can be Ior Q (SEQ ID NO: 87) (SEQ ID NO: 91) DX₁(S/T)QV, where X₁ can be F,EGDYSQV (SEQ ID NO: 93) EGDYSQVLEK (SEQ ID NO: 22) W, or Y (SEQ ID NO:92)

Compositions for detecting protein serine or threonine phosphataseactivity incorporate a motif (e.g., a recognition motif for a S/Tkinase) into a peptide, where one or more serine or threonine aminoacids in the motif are phosphorylated. Dephosphorylation of a serine orthreonine amino acid in the composition by a serine- orthreonine-directed protein phosphatase activity modulates the rate ofhydrolysis by a protease (e.g., chymotrypsin) as compared to thephosphorylated composition. Illustrative phosphatase motifs, recognitionmotifs, and peptides are set forth in Table 4, below, where (S/T)*indicates a phosphorylated serine or threonine, S* indicates aphosphorylated serine, and T* indicates a phosphorylated threonine.Table 4 TABLE 4 Illustrative Illustrative Recognition Peptide MotifMotif Sequence RRX₁(S/T)*L, where X₁ can be F, LRRFS*LG (SEQ ID NO: 96)ALRRFS*LGEK or Y (SEQ ID NO: 95) (SEQ ID NO: 97) LX₁(S/T)*TT, where X₁can be F, GLFS*TTP (SEQ ID NO: 99) RGGLFS*TTPGGTK W, or Y (SEQ ID NO:98) (SEQ ID NO: 100) X₁L(S/T)*LD, where X₁ can be F, DYLS*LDK (SEQ IDNO: 102) GDQDYLS*LDK W, or Y (SEQ ID NO: 101) (SEQ ID NO: 103)RX₁X₂(S/T)*X₃, where X₁ can be V, NRVFS*VA, (SEQ ID NO: 105)KLNRVFS*VAC, A, or Q, X₂ can be F, W, or Y, and PRAFS*VG, (SEQ ID NO:106) (SEQ ID NO: 108) X₃ can be V or L (SEQ ID NO: 104) RRQFS*LR (SEQ IDNO: 107) ARPRAFS*VGK, (SEQ ID NO: 109) RRRQFS*LRRKAK (SEQ ID NO: 110)TX₁S(S/T)*L, where X₁ can be F, W, RTFSS*LA (SEQ ID NO: 112)RPRTFSS*LAEGK or Y (SEQ ID NO: 111) (SEQ ID NO: 113) X₁X₂(S/T)*PX₃ whereX₁ can be P APFS*PGG, (SEQ ID NO: 115) VAPFS*PGGRAK, or I, X₃ can be F,W, Y, and HPFS*PKK, (SEQ ID NO: 116) (SEQ ID NO: 118) X₂ can be G, K, orD KIFS*PDV (SEQ ID NO: 117) AKHPFS*PKKAK, (SEQ ID NO: 114) (SEQ ID NO:119) IIKIFS*PDVEK, (SEQ ID NO: 120) X₁(S/T)*X₂X₃VA, where X₁ can beEFT*AYVA (SEQ ID NO: 122), DDEFT*AYVATRK, F, W, or Y, X₂ can be A, E, orQ, IFT*EYVA (SEQ ID NO: 123), (SEQ ID NO: 125) and X₃ can be Y or HVFT*QHVA (SEQ ID NO: 124) TGIFT*EYVATRK, (SEQ ID NO: 121) (SEQ ID NO:126) TGVFT*QHVATRK (SEQ ID NO: 127) IX₁(S/T)*IAN, where X₁ can be F,RIFS*IANS (SEQ ID NO: 129) QRIFS*IANSIVK W, or Y (SEQ ID NO: 128) (SEQID NO: 130) SIAX₁(S/T)*I, where X₁ can be F, DSIAFS*IV (SEQ ID NO: 132)RIDSIAFS*IVGK W, or Y (SEQ ID NO: 131) (SEQ ID NO: 133) (S/T)*VPPS*P(SEQ ID NO: 134) FS*VPPS*PD PRPFS*VPPS*PDK (SEQ ID NO: 135) (SEQ ID NO:136) DX₁X₂(S/T)*X₃, where X₁ can be A EDAFS*II, (SEQ ID NO: 138)EEDAFS*IIGK, or E, X₂ can be F, W, or Y, and X₃ EDEFS*QN (SEQ ID NO:139) (SEQ ID NO: 140) can be I or Q (SEQ ID NO: 137) REDEFS*QNIEEK (SEQID NO: 141) DX₁(S/T)*QV, where X₁ can be F, EGDYS*QV (SEQ ID NO: 143)EGDYS*QVLEK W, or Y (SEQ ID NO: 142) (SEQ ID NO: 144)Protease

Many proteases for use in the present invention are commonly availableat high purity. Typically, the proteolytic activity of a protease for acomposition is modulated by the presence or absence of apost-translationally modified (e.g., phosphorylated) amino acid in amotif. Preferred compositions exhibit a significant modulation, e.g. atleast 1.5, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50 or 100 foldmodulation, of proteolytic reactivity when modified as compared to whennon-modified. See Table 5 below for illustrative proteases. TABLE 5 NameEC Type Peptide Primary Specificity Caspase 3 Cysteine DXXD-P′₁ P₁ =Asp, P′₁ = neutral preferred Cathepsin G EC 3.4.21.20 Serine P₁-P′₁ P₁aromatic preferred, W, Y, F Chymotrypsin EC 3.4.21.1 Serine P₁-P′₁ P₁ =aromatic preferred, W, Y, F Elastase EC 3.4.21.36 Serine P₁-P′₁ P₁ =uncharged, non aromatic, e.g. A, V, L, 1, G, S, T Endoprotemase UnknownP₁-Asp P′₁ = Asp or P′₁ = Cysteic acid Asp-N P₁ = non-specificEndoproteinase EC 3.4.21.9 Serine Glu-P′₁ P₁ = Glu or Asp Glu-N P′₁ =non-specific Streptomyces EC 3.4.21.82 Serine Glu-P′₁ P₁ = Glu or Aspgriseus P′₁ = non-specific Staphylococcus EC 3.4.21.19 Serine Glu-P′₁ P₁= Glu or Asp aureus V8 P′₁ = non-specific

Proteases that may be used to measure peptide phosphorylation ordephosphorylation include those that recognize a composition thatincludes at least one motif position in which the presence or absence ofa phosphorylated amino acid modulates the activity of the proteasetowards that composition. The flexibility in choice of motifs containingor lacking phosphorylated amino acids (e.g., tyrosine, serine orthreonine) combined with the flexibility in choice of the proteaseenables many protein kinase or phosphatase activities to be measuredusing the present invention.

In a cell-based application of the present method, the expression of aprotease within a cell is regulated (e.g., using inducible nucleic acidconstructs that encode the protease). Suitable nucleic acid constructscan be designed and used as a matter of routine by those skilled in theart. In such cell-based assays, an appropriate measurable (e.g.,optical) property of a composition that includes at least one motifposition in which the presence or absence of a phosphorylated residuemodulates the activity of the protease towards that composition can bemonitored at one or more time intervals after the onset of increasedexpression of the protease.

Detectable Moieties

The choice of a detectable moiety is governed by a number of factorsincluding the mode of detection, the availability of specificinstrumentation, and the ease of coupling of the detectable moiety to apeptide. Other factors that may be relevant to a particular use includethe effect of a detectable moiety on the solubility of a composition,the kinetics of the post-translational activity or protease activitywith respect to a composition, and the desired detection sensitivity ofan assay.

Numerous detectable moieties are commercially available or can bereadily made. In general, a detectable moiety can exhibit an opticalproperty, a magnetic property, or a radioactive property. Thus, onceassociated with a peptide, a detectable moiety allows a resultingcomposition to exhibit an optical property, a magnetic property, or aradioactive property that is similar to or the same as that of thedetectable moiety alone. In some embodiments, the association of adetectable moiety with a peptide may alter a detectable property of thedetectable moiety to a greater or lesser extent. For example,conjugation of a fluorophore to a peptide may result in a compositionhaving an emission maximum that is different from that of thefluorophore alone in solution. In other embodiments, a detectable moietycan be a member of a specific binding pair. For example, a detectablemoiety can be the ligand member of a ligand-protein binding pair, e.g.,the biotin member of the biotin-streptavidin binding pair.

For fluorescent detectable moieties, preferred fluorophores typicallyexhibit good quantum yields, long excited state lifetimes, and largeextinction coefficients; are resistant to collisional quenching andbleaching; and should be easily conjugated to a peptide. Fluorophoresthat show absorbance and emission in the red and near-infrared range areuseful in whole animal studies because of reduced scattering backgroundfluorescence and greater transmission through tissues. Examples ofillustrative fluorophores include cyanines, oxazines, thiazines,porphyrins, phthalocyanines, fluorescent infrared-emitting polynucleararomatic hydrocarbons such as violanthrones, fluorescent proteins, nearIR squaraine dyes. (for example, as shown in Dyes and Pigments 17:19-27(1991), U.S. Pat. No. 5,631,169 to Lakowicz et al., issued May 20, 1997,and organo-metallic complexes such as ruthenium and lanthanide complexesof U.S. Pat. Nos. 4,745,076 and 4,670,572, the disclosures of which areincorporated herein by reference).

Suitable fluorophores and dark quenchers for use in the presentinvention are commercially available, e.g., from Molecular Probes(Eugene, Oreg.), Attotec (Germany), Amersham, and Biosearch Technologies(Novato, Calif.). Specific fluorophores include, without limitation,fluorescein isothiocyanate (especially fluorescein-5-isothiocyanate),5-FAM (5-carboxyfluorescein), 6-FAM (6-carboxyfluorescein), 5,6-FAM,7-hydroxycoumarin-3-carboxamide,6-chloro-7-hydroxycoumarin-3-carboxamide,dichlorotriazinylaminofluorescein, tetramethylrhodamine-5 (and-6)-isothiocyanate, 1,3-bis- (2-dialkylamino-5-thienyl)-substitutedsquarines, the succinimidyl esters of 5 (and 6) carboxyfluoroscein, 5(and 6)-carboxytetramethylrhodamine, and7-amino-4-methylcoumarin-3-acetic acid. Semiconductor fluorescentnanocrystals are available with a range of emission spectra, are highlyfluorescent and are also useful (see Bruchez et al., Science 281:2013-2016).

Lanthanide complexes (e.g., metal chelates of Eu or Th) are also usefuland have the advantage of not being quenched by oxygen. Their longlifetimes (on the order of ms as compared to ns for other fluorophores)may allow easy suppression of the auto-fluorescence of biologicalsamples, as fluorescent signals may be measured after background signalshave decayed. Accordingly, lanthanide complexes, such as Eu or Tb metalchelates, may be particularly useful, e.g., in time-resolved FRET(TR-FRET) applications. See, for example, U.S. Pat. Nos. 5,639,615,5,622,821, and 5,656,433.

Illustrative luminescent moieties include chemiluminescent,electroluminescent, and bioluminescent compounds. Preferredbioluminescent compounds include bioluminescent proteins such asfirefly, bacterial, or click beetle luciferases, aequorins, and otherphotoproteins (for example as described in U.S. Pat. No. 5,221,623,issued Jun. 22, 1989 to Thompson et al., U.S. Pat. No. 5,683,888 issuedNov. 4, 1997 to Campbell; U.S. Pat. No. 5,674,713 issued Sep. 7, 1997 toDeLuca et al.; U.S. Pat. No. 5,650,289 issued Jul. 22, 1997 to Wood; andU.S. Pat. No. 5,843,746 issued Dec. 1, 1998 to Tatsumi et al.).Preferred electroluminescent moieties include ruthenium complexes, asfor example described in U.S. Pat. No. 5,597,910 issued to Jan. 28, 1997to Gudibande. Preferred chemiluminescent moieties include those based on1,2-dioxetanes, as for example described in U.S. Pat. No. 4,372,745issued Feb. 8, 1983 to Mandle et al., U.S. Pat. No. 5,656,207 issuedAug. 12, 1997 to Woodhead et al., and U.S. Pat. No. 5,800,999 issuedSep. 1, 1998 issued to Bronstein et al.

Magnetic detectable moieties include MR contrast agents, e.g., chelatesof paramagnetic, ferromagnetic, or diamagnetic metal ions, or magneticparticles (e.g., USPIOs, MIONs; see U.S. Pat. No. 5,262,176). In someembodiments, a chelate may comprise a lipophilic group as described inU.S. Pat. No. 5,628,982, issued May 13, 1997 to Lauffer et al., and U.S.Pat. No. 5,242,681, issued Sep. 7, 1993 to Elgavish et al. For reviewsof metal chelates useful in MR imaging, see Lauffer, “Paramagnetic MetalComplexes as Water Proton Relaxation Agents for NMR Imaging: Theory andDesign,” Chem. Rev. 87(5):901-927 (1987); and Caravan et al.,“Gadolinium (III) Chelates as MRI Contrast Agents: Structure, Dynamics,and Applications,” Chem. Rev. 99(9):2293-2352 (1999).

In some applications it may be desirable to derivatize a detectablemoiety to render it more hydrophobic and permeable to cell membranes.The derivatizing groups may undergo hydrolysis inside cells toregenerate the compositions, thus trapping them within cells. For thispurpose, it is preferred that phenolic hydroxyls or free amines in thestructures are acylated with C₁-C₄ acyl groups (e.g. formyl, acetyl,n-butyl) or converted to, e.g., esters and carbonates, as described inBundgaard, H., Design of Prodrugs, Elsevier Science Publishers (1985),Chapter 1, page 3 et seq. Further modification of fluorescent moietiesmay also be accomplished e.g., as described in U.S. Pat. No. 5,741,657issued Apr. 21, 1998 to Tsien et al.

A detectable moiety may be attached to a peptide by a linker (L) thatprovides a spacer between the detectable moiety and the peptide, therebypreventing steric or charge interference of the detectable moiety on theinteraction between, e.g., the recognition motif of the peptide and akinase or phosphatase. Preferred linkers are substantially stable undercellular conditions and easily coupled to a peptide and detectablemoiety. Examples include flexible aliphatic linkers such asγ-amino-n-butyric acid (GABA), diaminopentane, and aminohexanoyl, aswell as rigid aromatic linkers. Such linkers are known in the art anddescribed for example in the Handbook of Fluorescent Probes and ResearchChemicals, by Richard Haugland, published by Molecular Probes.

Non-covalent methods of attachment may also be used to associate apeptide with a detectable moiety. For example, a peptide may be designedto encompass a specific binding site for a fluorescent moiety, asdescribed in pending U.S. Pat. Nos. 6,054,271; 6,008,378, and 5,932,474.Labeling may then be achieved by incubation of a peptide with amembrane-permeable fluorescent binding partner, which has the advantagesof enabling the expression of peptides within intact living cells, andthe subsequent labeling of these peptides in situ to create compositionsof the present invention within intact living cells.

Fluorescent Proteins

For some cell-based applications, fluorescent detectable moietiesinclude endogenously fluorescent proteins, functional engineeredfluorescent proteins, and variants thereof. Use of such proteins allowsthe fluorophore and peptide to be expressed within living cells withoutthe addition of other co-factors or fluorophores. Such compositionsprovide the ability to monitor post-translational activities withindefined cell populations, tissues, or a transgenic organism, forexample, by the use of inducible controlling nucleotide sequences toproduce a sudden increase in the expression of a composition andprotease.

Endogenously fluorescent proteins have been isolated and cloned from anumber of marine species including the sea pansies Renilla reniformis,R. kollikeri and R. mullerei and from the sea pens Ptilosarcus,Stylatula and Acanthoptilum, as well as from the Pacific Northwestjellyfish, Aequorea victoria (Szent-Gyorgyi et al. (SPIE conference1999), D. C. Prasher et al., Gene, 111:229-233 (1992)). These proteinsare capable of forming a highly fluorescent, intrinsic chromophorethrough the cyclization and oxidation of internal amino acids within theprotein that can be spectrally resolved from weakly fluorescent aminoacids such as tryptophan and tyrosine.

Fluorescent proteins have also been observed in other organisms. Forexample, the cloning and expression of yellow fluorescent protein fromVibrio fischeri strain Y-1 has been described by T. O. Baldwin et al.,Biochemistry (1990) 29:5509-15. This protein requires flavins asfluorescent co-factors. The cloning of Peridinin-chlorophyll a bindingprotein from the dinoflagellate Symbiodinium sp. was described by B. J.Moms et al., Plant Molecular Biology, (1994) 24:673:77. One usefulaspect of this protein is that it fluoresces in red. The cloning ofphycobiliproteins from marine cyanobacteria such as Synechococcus, e.g.,phycoerythrin and phycocyanin, is described in S. M. Wilbanks et al., J.Biol. Chem. (1993) 268:1226-35. These proteins require phycobilins asfluorescent co-factors, whose insertion into the proteins involvesauxiliary enzymes. The proteins fluoresce at yellow to red wavelengths.See also PCT US 01/04625.

A variety of mutants of the GFP from Aequorea victoria have been createdthat have distinct spectral properties, improved brightness, andenhanced expression and folding in mammalian cells compared to thenative GFP (e.g., see Table 7 of U.S. Pat. No. 6,410,255 and also GreenFluorescent Proteins, Chapter 2, pages 19 to 47, edited by Sullivan andKay, Academic Press; U.S. Pat. No. 5,625,048 to Tsien et al., issuedApr. 29, 1997; U.S. Pat. No. 5,777,079 to Tsien et al., issued Jul. 7,1998; and U.S. Pat. No. 5,804,387 to Cormack et al., issued Sep. 8,1998). In many cases these functional engineered fluorescent proteinshave superior spectral properties to wild-type Aequorea GFP and arepreferred for use in the compositions of the invention.

Cell-based Assays

The methods of the present invention can also be employed in cell-basedassays. Recombinant production of the compositions within living cellsinvolves, in one embodiment, expressing nucleic acids having sequencesthat encode a fluorescent protein (e.g., as a detectable moiety) and apeptide of interest as a fusion protein. In one embodiment, acomposition comprises a first fluorescent protein (e.g., as the firstdetectable moiety), a peptide containing a motif, such as a recognitionmotif for a Y or S/T kinase, a protease site, and a second fluorescentprotein (as a second detectable moiety) fused together as a singlepolypeptide chain. Nucleic acids encoding fluorescent proteins can beobtained by methods known in the art. For example, a nucleic acidencoding the protein can be isolated by polymerase chain reaction ofcDNA from a suitable organism using primers based on the DNA sequence ofthe fluorescent protein. PCR methods are described in, for example, U.S.Pat. No. 4,683,195; Mullis et al. (1987); Cold Spring Harbor Symp.Quant. Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press,NY, 1989). Suitable clones may then be identified, isolated andcharacterized by fluorescence activated cell sorting (FACS), typicallyenabling the analysis of a few thousand cells per second. Theconstruction of expression vectors and the expression of genes intransfected cells involve the use of molecular cloning techniques alsowell known in the art; see, e.g., Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds. Nucleic acids used to transfect cells with sequences codingfor expression of the polypeptide of interest generally will be in theform of an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thecomposition comprising the peptide and fluorescent proteins.

In an alternative embodiment, a composition can include a reporterprotein (e.g., as a first detectable moiety), a peptide containing amotif described herein, such as a recognition motif for a Y or S/Tkinase, a protease site, and a multimerized ubiquitin fusion proteintogether as a single polypeptide chain. In these embodiments, themotif-containing peptide functions as a linker between the reporterprotein and the multimerized ubiquitin fusion protein. Such apolypeptide can be used to carry out an assay for a kinase (or aphosphatase) in a cell. For example, if a suitable kinase is present andactive in a cell, the peptide will be phosphorylated and not subject todegradation by a protease, thereby allowing the ubiquitin fusion proteinto destabilize (e.g., promote degradation of) the reporter protein. Ifkinase activity is not present or is inhibited, the motif-containingpeptide will be subject to degradation by the protease, therebypreventing the multimerized ubiquitin fusion protein from destabilizingthe reporter protein and preserving reporter protein activity. Suitablereporter proteins, multimerized ubiquitin fusion proteins, andconstructs for use in such an embodiment are described in WO 01/57242.

Methods of Measurement and Detection

Methods of measurement and detection include, without limitation,fluorescence spectroscopy, luminescence spectroscopy, absorptionspectroscopy, and magnetic resonance spectroscopy (e.g., NMR, MRI).Fluorescent methods include continuous or time resolved fluorescencespectroscopy, fluorescence correlation spectroscopy, fluorescencepolarization spectroscopy, and resonance energy based fluorescencespectroscopy. Methods of performing such assays on fluorescent materialsare well known in the art and are described in, e.g., Lakowicz, J. R.,Topics in Fluorescence Spectroscopy, volumes 1 to 3, New York: PlenumPress (1991); Herman, B., Resonance energy transfer microscopy, inFluorescence Microscopy of Living Cells in Culture, Part B, Methods inCell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego:Academic Press (1989), pp. 219-243; Turro, N. J., Modern MolecularPhotochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.(1978), pp. 296-361; and Bernard Valeur, “Molecular Fluorescence:Principles and Applications” Wiley VCH, 2002.

The selection and use of specific detectable moieties (e.g., specificfluorophores or quenchers) for particular applications is generallyknown in the art; for example, see Berlman, I. B., Energy transferparameters of aromatic compounds, Academic Press, New York and London(1973), which contains tables of spectral overlap integrals for theselection of resonance energy transfer partners. Additional informationsources include the Molecular Probes Catalog (2003) and website; andTsien et al., 1990 Handbook of Biological Confocal Microscopy, pp.169-178.

Methods and Assays

Compositions of the present invention can be used in a variety ofmethods. Standard techniques are usually used for chemical synthesis,fluorescence monitoring and detection, optics, molecular biology, andcomputer software and integration. Chemical reactions, cell assays, andenzymatic reactions are typically performed according to themanufacturer's specifications where appropriate. See, generally,Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes) NewYork: Plenum Press (1991), and Lakowicz, J. R. Emerging applications offlorescence spectroscopy to cellular imaging: lifetime imaging,metal-ligand probes, multi photon excitation and light quenching,Scanning Microsc. Suppl. Vol. 10 (1996) pages 213-24, for fluorescencetechniques; Sambrook et al., Molecular Cloning: A Laboratory Manual,2ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., for molecular biology methods; Cells: A Laboratory Manual, 1^(st)edition (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., for cell biology methods; and Optics Guide 5 Melles Griot® IrvineCalif., and Optical Waveguide Theory, Snyder & Love (published byChapman & Hall) for general optical methods, all of which areincorporated herein by reference.

Compositions of the present invention can be used to preparephosphorylated compositions. Methods of the present invention can alsobe used to characterize a kinase or a phosphatase, e.g., to measurekinetic or thermodynamic parameters. In one method, a composition ofmatter is used in a reaction with a kinase or phosphatase. Thecomposition is contacted with a kinase or phosphatase under conditionseffection for the kinase or phosphatase to phosphorylate ordephosphorylate the composition, respectively, and the ability of thekinase to phosphorylate (or the phosphatase to dephosphorylate) thecomposition is measured. Ability to phosphorylate a composition may bemeasured in a number of ways, e.g., in terms of % phosphorylation of thecomposition in a given time period, at a particular concentration ofkinase, or at a particular temperature; or in terms of kineticparameters (e.g., V_(max), K_(m)). See Examples 1 and 2 and FIGS. 3-5and 8-9. Methods for using a composition are described in, for example,U.S. Pat. Nos. 6,410,255, 5,917,012, and in Rodems et al., “A FRET-basedAssay is Platform for Ultra-High Density Drug Screening of ProteinKinases and Phosphatases,” ASSAY and Drug Development Technologies, Vol.1 (1-1), November 2002.

Methods of the present invention can be used to determine whether or nota composition of matter is a substrate for a kinase or phosphatase. Inone method, a composition of matter is contacted with an enzyme, e.g., aprotein kinase or protein phosphatase; the composition and enzyme arethen contacted with a protease; and a measurable property in theprotease mixture is monitored. A measurable property can be a detectableproperty of a composition, a detectable property of a cleavage productof a composition (e.g., a detectable property of a donor fluorescentmoiety or a detectable property of an acceptor fluorescent moiety), adetectable property of an enzyme, buffer, or reagent, or any combinationthereof. For example, a measurable property may be the net fluorescenceemission at a wavelength (or a ratio of the net fluorescence emission attwo wavelengths) after a composition has been partially cleaved (e.g.,70% cleavage). In this situation, the measurable property reflects thecontribution of the intact composition and the mixture of cleavageproducts to the fluorescence emission of the mixture at the particularwavelength under consideration.

For kinase reactions, ATP is generally included when a composition iscontacted with kinase (e.g., during an incubation with the kinaseenzyme). As one of skill in the art will recognize, in those methodsemploying phosphatase enzymes, a phosphorylated composition of matter asdescribed above is contacted with a phosphatase enzyme. Incubationconditions for a contacting step can vary, e.g., in enzymeconcentration, substrate concentration, temperature, and length of time.Incubation temperature conditions typically can be from about 15 toabout 40° C.; in some embodiments, the temperature may be about roomtemperature, e.g., about 20-25° C.

A measurable property in a protease mixture may be compared to ameasurable property in a control mixture. A control mixture can includethe composition of matter and the protease and is typically preparedwithout the addition of enzyme and/or without the addition of ATP (e.g.,for kinase reactions). Other control samples can comprise aphosphorylated version of the composition incubated with the protease inorder to correct for any cleavage of the phosphorylated composition bythe protease. One of skill in the art can typically design appropriatecontrol mixtures for reference.

A measurable property can be monitored during an incubation with akinase or phosphatase or when a kinase or phosphatase incubation iscomplete. Similarly, a measurable property can be monitored during aprotease incubation or when a protease incubation is complete.Typically, a measurable property is measured after a predetermined timeperiod of a kinase, phosphatase, or protease incubation. For example, ameasurable property may be measured within 12 hours of the initiation ofa kinase (phosphatase) or protease incubation. In some embodiments, ameasurable property is measured within 30 minutes, 1 hour, 2 hours, or 4hours of initiation. A protease incubation can be stopped by a number ofknown methods, including the addition of a reagent to inhibitproteolytic activity (e.g., aprotinin, PMSF, TPCK, AEBSF, chymotrypsininhibitor 1, chymotrypsin inhibitor 2), by heating and/or denaturing theprotease sample, and by altering pH or metal concentration (e.g., bychelating an active site metal).

A composition is identified as a substrate of a kinase (or phosphatase)if a measurable property in the protease mixture is different from themeasurable property in the appropriate control mixture. Generally, themeasurable property should be statistically significantly different fromthe measurable property in the control mixture. As one of skill in theart will recognize, whether or not a difference is statisticallysignificant will depend on the type of measurable property, the type ofmeasurement, and the experimental conditions. It is understood that whencomparing measurable properties, a statistically significant differenceindicates that that substrate may warrant further study. Typically, adifference in measurable properties is considered statisticallysignificant at p<0.05 with an appropriate parametric or non-parametricstatistic, e.g., Chi-square test, Student's t-test, Mann-Whitney test,or F-test. In some embodiments, a difference is statisticallysignificant at p<0.01, p<0.005, or p<0.001.

Typically, a detectable property will be an optical property, such as afluorescence property. In one aspect, the method may be based on adifference in a fluorescence anisotropy measurement of a compositionbefore and after cleavage with a protease. In this case, a compositiontypically comprises a peptide moiety which contains a motif, e.g., arecognition motif for a kinase or phosphatase, a protease site, and afluorescent detectable moiety. Modification of the peptide by the kinase(or phosphatase) activity results in a modulation of the rate at which aprotease cleaves the peptide, which is sensed by a measurable (e.g.,statistically different) change in fluorescence polarization of thecomposition upon cleavage.

Polarization measurements are based on the relative rotational movementof a fluorophore compared to the excited state life-time of thatfluorophore. For globular molecules in dilute solution, the relationshipbetween polarization (p) and the degree of rotational movement can bereadily derived (see Weber, Polarization of the fluorescence ofsolutions, in Fluorescence and Phosphorescence Analysis, Don Hercules(ed.), Interscience Publishers, New York, Chapter 8, pages 217-240(1966)). Rotational movement can be related to the rotational diffusionconstant of the molecule, and hence to the molecular volume. In practicethere is a close correlation between molecular size and relativepolarization of emitted light from a fluorophore. As a consequence, asignificant change in fluorescence polarization can occur whencompositions of the present invention are acted upon by a protease.Polarization-based assays are relatively easy to set up and can beobtained over a wide concentration, temperature, and ionic strengthrange.

In another embodiment of the method, fluorescence anisotropymeasurements can be enhanced by attaching one end of a peptide of acomposition to a solid matrix or a bead. In either case, cleavage of thecomposition results in a large drop in fluorescence polarization becauseof the increased rotational flexibility of the cleavage product of thecomposition once separated from the solid matrix or bead.

In another aspect, the present invention takes advantage of resonanceenergy transfer either between two fluorescent moieties (FRET), or abioluminescent moiety and fluorescent moiety (bioluminescent resonanceenergy transfer, BRET), or a fluorescent moiety and a quencher (e.g.,RET dark quenching) to provide a fluorescent readout.

In FRET applications, a composition typically comprises a firstfluorescent detectable moiety and a second fluorescent detectable moietycoupled to a peptide such that a motif (e.g., a recognition motif) and aprotease cleavage site are located between the two detectable moieties.In this case, cleavage of the peptide by a protease results in analteration in energy transfer between the first fluorescent moiety andthe second fluorescent moiety that may be used to monitor and measurekinase or phosphatase activity.

In FRET cases, fluorescent moieties are typically chosen such that theexcitation spectrum of one of the moieties (the acceptor fluorescentmoiety) overlaps with the emission spectrum of the donor fluorescentmoiety. The donor fluorescent moiety is excited by light of appropriatewavelength and intensity within the donor fluorescent moiety'sexcitation spectrum and under conditions in which direct excitation ofthe acceptor fluorophore is minimized. The donor fluorescent moiety thentransfers the absorbed energy by non-radiative means to the acceptor,which subsequently re-emits some of the absorbed energy as fluorescenceemission at a characteristic wavelength. FRET applications can includeTR-FRET applications. In these embodiments, a Ln complex, such as a Euor Tb metal chelate, is used as a fluorescent donor moiety, as describedabove. Typically, the Ln complex is chosen so that one of its emissionbands overlaps with an excitation band of the acceptor fluorescentmoiety.

FRET can be manifested as a reduction in the intensity of thefluorescent signal from the donor, reduction in the lifetime of itsexcited state, and/or an increase in emission of fluorescence from theacceptor fluorescent moiety. When a peptide having a donor fluorescentmoiety and acceptor fluorescent moiety is cleaved, the donor fluorescentmoiety and the acceptor fluorescent moiety physically separate, and FRETis diminished or eliminated. Under these circumstances, fluorescenceemission from the donor increases and fluorescence emission from theacceptor decreases. Accordingly, a ratio of emission amplitudes atwavelengths characteristic (e.g., the emission maximum) of the donorrelative to the acceptor should increase as compared to the same ratiounder FRET conditions (e.g., when emission of the donor is quenched(e.g., reduced) by the acceptor).

The efficiency of FRET is dependent on the separation distance and theorientation of the donor fluorescent moiety and acceptor fluorescentmoiety, the fluorescent quantum yield of the donor moiety, and thespectral overlap with the acceptor moiety. Forster derived therelationship:E=(F ^(o) −F)/F ^(o) =Ro ⁶/(R ⁶ +Ro ⁶)where E is the efficiency of FRET, F and F^(o) are the fluorescenceintensities of the donor in the presence and absence of the acceptor,respectively, and R is the distance between the donor and the acceptor.Ro, the distance at which the energy transfer efficiency is 50% ofmaximum is given (in Å) by:Ro=9.79×10³(K ² QJn ⁻⁴)^(1/6)where K² is an orientation factor having an average value close to 0.67for freely mobile donors and acceptors, Q is the quantum yield of theunquenched fluorescent donor, n is the refractive index of theintervening medium, and J is the overlap integral, which expresses inquantitative terms the degree of spectral overlap. The characteristicdistance Ro at which FRET is 50% efficient depends on the quantum yieldof the donor, the extinction coefficient of the acceptor, the overlapbetween the donor's emission spectrum and the acceptor's excitationspectrum, and the orientation factor between the two fluorophores.

Changes in the degree of FRET can be determined as a function of achange in a ratio of the amount of fluorescence from the donor andacceptor moieties, a process referred to as “ratioing.” By calculating aratio, the assay is less sensitive to, for example, well-to-wellfluctuations in substrate concentration, photobleaching and excitationintensity, thus making the assay more robust. This is of particularimportance in automated screening applications where the quality of thedata produced is important for its subsequent analysis andinterpretation.

For example, in some embodiments of the method, a ratiometric analysisis performed, wherein a ratio of fluorescence emission at two differentwavelengths is compared between a protease mixture and a controlmixture. In a typical FRET-based assay, the two wavelengths cancorrespond to the emissions maxima for two detectable (e.g.,fluorescent) moieties of the composition. Thus, if a composition is asubstrate for a kinase, the phosphorylated composition will be lesssusceptible to cleavage by a protease. Accordingly, the phosphorylatedcomposition will maintain FRET between the donor and acceptor moieties(e.g., the FRET pair), resulting in a low emissions ratio of the donorto the acceptor moiety. A control sample in such a case, however, willbe subject to cleavage by the protease. Cleavage disrupts FRET betweenthe donor and acceptor moieties, leading to a larger emissions ratio ofthe donor to the acceptor moiety. In some embodiments, the emissionsratio of the control mixture will be more than 1.5, 2, 3, 4, 5, 7, 10,15, 20, 25, 30, 40, 50, or 100 times larger than the emissions ratio ofa protease mixture.

The present invention can also be used to determine whether a sample(e.g., a cell, an extract, a purified protein, a tissue, an organism)has general kinase or phosphatase activity or a specific kinase orspecific phosphatase activity, e.g., abl-1 kinase activity. The methodtypically involves contacting a sample with a composition of matter(e.g., under appropriate conditions to enable phosphorylation (ordephosphorylation) of the composition), and then contacting the sampleand composition mixture with a protease, e.g., a protease known tocleave the composition in the absence of the post-translationalmodification. The degree of post-translational modification activity inthe sample is assessed, e.g., as described above, such as by monitoringa measurable property of the sample-composition mixture and comparing itto the measurable property of a control mixture.

In some cases, a composition and a protease may be added to a sample atthe same time. Alternatively, in the case where a sample contains cells,the method would typically involve stimulation of the cells and theneither lysing the cells in the presence of the composition or, in thecase where the composition is expressed within the cells, lysing thecells in the presence of a protease to measure composition modification.

One application of the assay is to either introduce or express thecomposition in living eukaryotic or prokaryotic cells to enable themeasurement of intracellular post-translational activities. In oneaspect, the method would involve a composition comprising a firstfluorescent protein, a peptide containing a motif (e.g., a recognitionmotif) and a protease site, and a second fluorescent protein fusedtogether as a single polypeptide chain. In this case the firstfluorescent protein and the second fluorescent protein would be selectedto enable FRET to occur as described above. A pair of functionalengineered fluorescent proteins for example would be, Topaz (S65G, S72A,K79R, T203Y) and W1B (F64L, S65T, Y66W, N1461, M153T,V163A), as shown inTable 7 of U.S. Pat. No. 6,410,255.

In another aspect, a method can involve a composition comprising apeptide containing one or more binding sites for a fluorescent moiety, amotif (e.g., a recognition motif), and a protease cleavage site. Forexample, a binding site could comprise a sequence that recognizes afluorescent moiety, as described in pending U.S. patent application Ser.No. 08/955,050, filed Oct, 21, 1997, entitled Methods of Using SyntheticMolecules and Target Sequences; Ser. No. 08/955,859, filed Oct. 21,1997, entitled Synthetic Molecules that Specifically React with TargetSequences; and Ser. No. 08/955,206, filed Oct. 21, 1997, entitled TargetSequences for Synthetic Molecules. In this case, expression of a peptidecomprising a recognition motif, protease cleavage site, and binding sitecould be accomplished using genetic means as described above. Theaddition of a membrane-permeable fluorescent moiety capable of bindingto the binding site would enable the creation in situ of compositionaccording to the present invention.

Another application of the method is to use inducible controllingnucleotide sequences to produce a sudden increase in the expression ofeither a composition, a kinase or phosphatase, or a protease, e.g., byinducing expression of a construct. A suitable protease could beexpressed within a cell, or induced, or introduced using amembrane-translocating sequence (see U.S. Pat. No. 5,807,746, issuedSep. 15, 1998 to Lin et al.) The efficiency of FRET may be typicallymonitored at one or more time intervals after the onset of increasedexpression of a protease.

In BRET applications, a composition typically comprises a luminescentmoiety and a fluorescent moiety coupled to a peptide such that a motifand protease site are located between them. In this case, cleavage ofthe peptide by a protease results in an alteration in energy transferbetween the luminescent moiety and the fluorescent moiety that may beused to determine kinase or phosphatase activity, as described above. Inthis case, the luminescent and fluorescent moieties are typically chosensuch that the emission spectrum of the luminescent moiety overlaps withthe excitation spectrum of the fluorescent moiety. Because a luminescentmoiety provides light through a chemiluminescent, electroluminescent, orbioluminescent reaction, there is no requirement for direct lightexcitation to create the excited state in the luminescent moiety.Instead, appropriate substrates or a voltage must be provided to (orapplied to) the luminescent moiety to create an excited stateluminescent moiety. In the case of bioluminescent proteins, suchsubstrates are generically referred to as luciferins (for example, seeU.S. Pat. No. 5,650,289 issued Jul. 22, 1997 to Wood). If BRET occurs,the energy from the excited state of a luminescent moiety is transferredto a fluorescent moiety by non-radiative means, rather than beingemitted as light from the luminescent moiety. Because luminescent andfluorescent moieties typically are chosen to emit light atcharacteristic wavelengths, an emission ratio of the two can alsoprovide a ratiometric readout as described for the FRET basedapplications above. BRET can be manifested as a reduction in theintensity of a fluorescent signal from the luminescent moiety, areduction in the lifetime of its excited state, and/or an increase inemission of fluorescence from the fluorescent moiety. When a peptidesubstrate that connects a luminescent moiety and a fluorescent moiety iscleaved, the luminescent moiety and the fluorescent moiety physicallyseparate, and BRET is diminished or eliminated. Under thesecircumstances, light emission from the luminescent moiety increases andfluorescence emission from the fluorescent moiety decreases. Theefficiency of BRET is typically dependent on the same separation andorientation factors as described above for FRET.

In dark quenching RET applications, a composition typically comprises afirst fluorescent moiety (e.g., a donor) and a dark quencher moiety(e.g., acceptor) coupled to the peptide such that a motif and proteasesite are located between them. In this case, cleavage of the peptide bya protease results in an alteration in energy transfer between the firstfluorescent moiety and the dark quencher moiety that may be used tomonitor post-translational activity. A fluorescent moiety and darkquencher moiety are typically chosen such that the absorption spectrumof the dark quencher (the acceptor moiety) overlaps with the emissionspectrum of the donor fluorescent moiety. The donor fluorescent moietyis excited by light of appropriate intensity within the donorfluorescent moiety's excitation spectrum. The donor fluorescent moietythen transfers the absorbed energy by non-radiative means to the darkquencher, which in this case does not re-emit a substantial amount ofthe absorbed energy as light (e.g., forming a dark quenching RET pair).Dark quenching RET can be manifested as a reduction in the intensity ofa fluorescent signal from a donor or a reduction in the lifetime of itsexcited state. When a peptide that connects a donor fluorescent moietyand a dark quencher moiety is cleaved, the donor fluorescent moiety andthe dark quencher moiety physically separate, and dark quenching RET isdiminished or eliminated. Under these circumstances, fluorescenceemission from the donor fluorescent moiety increases.

Another mechanism of quenching contemplated in the present inventioninvolves the formation and detection of an excitonic dimer (e.g., staticquenching) between a fluorophore and a quencher. Typically, staticquenching results when the interaction of a fluorophore with a quencherforms a stable non-fluorescent or weakly fluorescent ground statecomplex. Since this complex typically has a different absorptionspectrum from the fluorophore, the presence of an absorption change isdiagnostic of this type of quenching (in contrast, collisional quenchingis a transient excited state interaction and therefore does not affectthe absorption spectrum). Pure static quenching can reduce the intensityof fluorescence but does not necessarily decrease the measured lifetimeof emission.

In magnetic detection-based assays, a composition typically comprises ametal chelate or metal particle, as described above, coupled to apeptide having a motif (e.g., a recognition motif) and a protease site.In these cases, the metal chelate or particle should be chosen to have acharacteristic magnetic signal, e.g., T₁, T₂, or R₁, that will changewhen it is bound or associated with the intact peptide as compared tothe cleaved peptide. Cleavage of the peptide by a protease results in analteration in the magnetic signal that may be used to monitorpost-translational activity.

In radio-isotope detection-based assays, a composition typicallycomprises a radioisotope (e.g., a radiolabel such as ³²P, ³⁵S, ³H, ¹⁴Cor others known to those of skill in the art) coupled to a peptidehaving a motif (e.g., a recognition motif) and a protease site. In thesecases, monitoring of a location of a radiolabel (e.g., in a gel) ormonitoring of a size of a cleavage product (e.g., in a gel) before andafter proteolytic cleavage provides a method for monitoringpost-translational activity.

The assays of the present invention can be used in drug screening assaysto identify compounds that alter or modulate a kinase or phosphataseactivity. In one embodiment, an assay is performed on a sample in vitro(e.g. in a sample isolated from a cell, or a cell lysate, or a purifiedor partially-purified enzyme) containing an activity for which a drugscreen is desired. A sample containing a known amount of activity iscontacted with a composition of the invention and with a test compound.The activity of the sample is monitored after addition of a protease, asdescribed above, for example, by monitoring a measurable property of thecomposition. A measurable property of the sample in the presence of thetest compound can be compared with the measurable property of a samplesimilarly treated in the absence of the test compound (e.g., the controlreaction). A difference indicates that the test compound alters theactivity. In preferred embodiments, the method is used to evaluateputative inhibitors of a kinase or phosphatase activity.

In another embodiment, an ability of a test compound to alter or tomodulate a kinase or phosphatase activity in a cell-based assay may bedetermined. In these assays, cells transfected with an expression vectorencoding a composition of the invention, as described above, are exposedto different amounts of the test compound, and the effect on ameasurable property (e.g., an optical property such as FRET orfluorescence polarization) in each cell can be determined afterinduction or introduction of a suitable protease. Typically, as with anymethod of the present invention, the change in the measurable propertyis compared to that of untreated controls.

Any of the methods of the present invention can be modified to beperformed in a high-throughput or ultra-high-throughput manner. Forexample, a method to identify a substrate of a particular kinase orphosphatase may be modified to contact a plurality of compositions(e.g., two or more different compositions), independently, with aparticular kinase or phosphatase enzyme, to form a plurality of enzymemixtures. Each enzyme mixture is treated with a protease, and ameasurable property of each protease mixture is monitored and comparedto an appropriate control sample. Similarly, a particular compositioncan be evaluated for its suitability as a substrate of a plurality ofkinases or phosphatases (e.g., two or more different kinases orphosphatases). Thus, a particular composition of matter may becontacted, independently, with a plurality of enzymes to form aplurality of enzyme mixtures. Each mixture is treated with a proteaseand a measurable property of each protease mixture is monitored andcompared to an appropriate control sample. As one of skill in the artwill appreciate, such high-throughput methods are particularly amenableto multi-well plate or 2-D array panel formats, wherein a plurality ofcompositions are screened for suitability as substrates for a pluralityof different enzymes. See Example 4, below. Devices for incubating andmonitoring multi-well plates are known in the art. Similar panel assaysmay be envisioned for methods to identify modulators of a kinase orphosphatase activity. See Example 3, below.

In another embodiment, a plurality of different compositions of mattermay be contacted simultaneously with a single kinase or phosphatase; thereaction mixture may then be contacted with a protease; and a pluralityof measurable properties may be monitored and compared to the measurableproperties of an appropriate control sample. Typically, each of thecompositions of matter comprises a FRET pair having a characteristicexcitation wavelength of the donor and emissions ratio of the donor tothe acceptor, so that each FRET pair can be tracked independently of theothers. An appropriate control sample would include the plurality ofcompositions of matter treated with the protease in the absence of thekinase, phosphatase, and/or ATP. As one of skill in the art willrecognize, other measurable properties can be similarly monitored tofacilitate the use of such a method with detectable moieties for darkquenching RET and magnetic detection applications.

Alternatively, arrays of compositions having known recognition motifsmay be created in order to create an activity profile of kinase orphosphatase activities in a sample. In this case, screening of the arrayis used to characterize the activities within a sample by incubating thearray with a sample containing the activities, adding an appropriateprotease, and then monitoring a measurable property from each member ofthe array. Those array members that are more efficiently modified afterexposure to the sample may be identified by the degree to which themeasurable property of that array member is altered as compared to theappropriate control samples.

The dynamic range, quality, and robustness of the methods of the presentinvention can be evaluated statistically. For example, the Z′-Factor isa statistic designed to reflect both assay signal dynamic range and thevariation associated with signal measurements. Signal-to-noise (S/N) orsignal-to-background (S/B) ratios alone are unsatisfactory in thisregard because they do not take into account the variability in sampleand background measurements and signal dynamic range. The Z′-Factortakes into account these factors, and because it is dimensionless, itcan be used to compare similar assays. The relationship of Z′-factorvalues to assay quality are summarized in Table 6, below. Typically,assays of the present invention yield Z′-factors of greater than orequal to 0.5.

A Z′-factor may be determined by evaluating the dynamic range of amethod. In some embodiments, the dynamic range may be defined by 0%inhibition and 100% inhibition controls. A 0% inhibition control isperformed by contacting a composition of the present invention with akinase and ATP to form a kinase mixture, contacting the kinase mixturewith a protease to form a protease mixture, and monitoring a measurableproperty of the protease mixture. A measurable property can be anemissions ratio, such as the ratio of coumarin emission at 445 nm tofluorescein emission at 520 nm. Typically, the reaction conditions ofthe kinase reaction are chosen to phosphorylate about 10-40% of thecomposition in a predetermined time period (e.g., 30 mins., 1 hr., 2hr., or 4 hr.). The % phosphorylation of a sample can be calculated byusing the following equation:% Phosphorylation=1−[((Emission Ratio×Fc)−Fa)/((Fb−Fa)+(EmissionRatio×(Fc−Fd)))]where Emission Ratio is the ratio of donor emission signal to acceptoremission signal, as indicated above; Fa is the average donor emissionsignal of the 100% phosphorylation control; Fb is the average donoremission signal of the 0% phosphorylation control; Fc is the averageacceptor emission signal of the 100% phosphorylation control; and Fd isthe average acceptor emission signal of the 0% phosphorylation control.

The 100% inhibition control is performed similarly, but in the absenceof ATP (100% inhibition of the kinase), to yield 0% phosphorylatedcomposition. A 100% phosphorylated composition can also be included as acontrol. Both 0% and 100% inhibition controls can be performed induplicate. The Z′-factor is then calculated as follows:Z′-factor=(1−(3×σ0% inhibition)+(3×σ100% inhibition))/(μ of 100%inhibition−μ of 0% inhibition)

TABLE 6 Z′-factor value Relation to Assay Quality  1 Excellent Assay 1 >Z′ ≧ 0.5 An excellent assay 0.5 > Z′ > 0 A double assay  0 A “yes/no”type assay <0 Assay unreliable

The methods of the present invention can be used with various systemsfor spectroscopic measurement. In one embodiment, the systemcomprises 1) a reagent for an assay and 2) a device comprising at leastone plate (e.g., a multi-well plate) or container and a platform, suchas a multi-well plate platform, e.g., for incubating and/or detecting asignal from the plate or container. The system can further comprise adetector, such as a detector appropriate for detecting a signal from asample or a plate. The system can comprise multiple plates or containersor multi-well platforms. In this context, a reagent for an assayincludes any reagent useful to perform biochemical or biological invitro or in vivo testing procedures, such as, for example, buffers,co-factors, proteins such as enzymes or proteases, carbohydrates,lipids, nucleic acids, active fragments thereof, organic solvents suchas DMSO, chemicals (e.g., ATP), analytes, therapeutics, compositions,cells, antibodies, ligands, and the like. In this context, an activefragment is a portion of a reagent that has substantially the activityof the parent reagent.

The compositions of the present invention are suited for use withsystems and methods that utilize automated and integratable workstationsfor identifying substrates and modulators of kinase or phosphataseactivity. Such systems are described generally in the art (see U.S. Pat.No. 4,000,976 to Kramer et al. (issued Jan. 4, 1977), U.S. Pat. No.5,104,621 to Host et al. (issued Apr. 14, 1992), U.S. Pat. No. 5,125,748to Bjornson et al. (issued Jun. 30, 1992), U.S. Pat. No. 5,139,744 toKowalski (issued Aug. 18, 1992), U.S. Pat. No. 5,206,568 to Bjornson etal. (issued Apr. 27, 1993), U.S. Pat. No. 5,350,564 to Mazza et al.(Sep. 27, 1994), U.S. Pat. No. 5,589,351 to Harootunian (issued Dec. 31,1996), and PCT Application Nos. WO 93/20612 to Baxter Deutschland GMBH(published Oct. 14, 1993), WO 96/05488 to McNeil et al. (published Feb.22, 1996), WO 93/13423 to Agong et al. (published Jul. 8, 1993) andPCT/US98/09526 to Stylli et al., filed May 14, 1998).

For some embodiments of the invention, particularly for plates with 96,192, 384 ,864 and 3456 wells per plate, detectors are available forintegration into the system. Such detectors are described in U.S. Pat.No. 5,589,351 (Harootunian), U.S. Pat. No. 5,355,215 (Schroeder), andPCT patent application WO 93/13423 (Akong). Alternatively, an entireplate may be “read” using an imager, such as a Molecular DynamicsFluorImager 595 (Sunnyvale, Calif.). Multi-well platforms having greaterthan 864 wells, including 3,456 wells, can also be used in the presentinvention (see, for example, PCT Application PCT/US98/11061, filed Jun.2, 1998).

In another embodiment, the system may comprise a two dimensional arrayof compositions dispersed on a substratum (e.g., a multi-well plate),for example as described in U.S. Pat. No. 4,216,245 issued Aug. 5, 1980to Johnson, U.S. Pat. No. 5,721,435 issued Feb. 24, 1998 to Troll, andU.S. Pat. No. 5,601,980 issued Feb. 11, 1997 issued to Gordon et al.Such a system provides the ability to rapidly profile large numbers ofcompositions and or large numbers of samples in a simple, miniaturizedhigh throughput format.

The present invention also provides articles of manufacture, such askits. Typically, a kit includes a container and a composition of matterof the present invention. In some embodiments, a kit can include one ormore of the following: a multi-well plate, a protease, one or moreenzymes (kinase or phosphatase enzymes), buffers, a source of ATP, anddirections for use of the kit. A kit can be useful for determiningsubstrates of kinase or phosphatase activity or for identifying amodulator of a kinase or phosphatase activity.

EXAMPLES Example 1 Determination of Kinase Kinetic Parameters withKinase Substrates

a. Determination of Kinetic Parameters for Akt 1 Kinase, aSerine/threonine Kinase

ATP was serially diluted in a Coming 384-well round bottom non-bindingsurface plate. A substrate for Akt 1, SEQ ID NO: 59 modified by having a7-hydroxycoumarin moiety conjugated to the c-NH2 of the C-terminallysine and a 5-FAM moiety conjugated to an N-terminal GABA linker,prepared as described in U.S. Pat. No. 6,410,255, was mixed with Akt 1kinase and added to the ATP dilutions. The final concentration of SEQ IDNO: 59 was 2 μM in each well; the final concentration of Akt1 was 16 nM.The plates were allowed to incubate at room temperature for 1 hour.Chymotrypsin was then added to each well (final concentration 100 ng/ml)and the plates allowed to incubate for 1 hour at room temperature. Theplate was read using a TECAN SAFIRE™ monochromator-based fluorescenceplate reader (Excitation at 400 nm (12 nm bandwidth); Emission at 445 nm(12 nm bandwidth); and Emission at 520 nm (12 nm bandwidth). Curvefitting and data presentation were performed using Prism™ software fromGraphPad Software, Inc. The apparent Km was 15 μM.

b. Determination of Kinetic Parameters for Abl 1 Kinase, a TyrosineKinase

ATP was serially diluted (0.8 μM to 100 μM) in a Corning 384-well roundbottom non-binding surface plate. A substrate for Abl 1, SEQ ID NO: 3modified by having a 7-hydroxycoumarin moiety conjugated to the ε-NH2 ofthe C-terminal lysine and a 5-FAM moiety conjugated to an N-terminalGABA linker, prepared as described in U.S. Pat. No. 6,410,255, was mixedwith Abl 1 kinase and added to the ATP dilutions. The finalconcentration of SEQ ID NO: 3 was 2 μM in each well; the finalconcentration of Abl-1 was 16 nM. The plates were allowed to incubate atroom temperature for 1 hour. Chymotrypsin was then added to each well(final concentration 1.25 ng/ml) and the plates allowed to incubate for1 hour at room temperature. The plate was read using a TECAN SAFIRE™monochromator-based fluorescence plate reader (Excitation at 400 nm (12nm bandwidth); Emission at 445 nm (12 nm bandwidth); and Emission at 520nm (12 nm bandwidth). Curve fitting and data presentation were performedusing Prism™ software from GraphPad Software, Inc. As can be seen fromFIG. 3, the Vmax of Abl 1 kinase with SEQ ID NO: 3 is 7%, correspondingto 0.02 pmol/min, and the apparent Km is 7 μM. See FIG. 3.

Example 2 Dependence of Percent Phosphorylation on Kinase Concentrationand Evaluation of Z′-Factor Values

a. Akt 1

Akt 1 kinase was serially diluted in a 384 well plate (finalconcentration ranging from 156 ng/ml to 20,000 ng/ml). Kinase reactionswere initiated by the addition of a solution of Akt 1 substrate, SEQ IDNO: 59 (modified as described in Example la above; 2 μM finalconcentration), and ATP (15 μM final concentration). The plates wereallowed to incubate at room temperature for 1 hour. Chymotrypsin wasthen added to each well (final concentration 100 ng/ml) and the platesallowed to incubate for 1 hour at room temperature. The plate was readusing a TECAN SAFIRE™ monochromator-based fluorescence plate reader(Excitation at 400 nm (12 nm bandwidth); Emission at 445 nm (12 nmbandwidth); and Emission at 520 nm (12 nm bandwidth). Curve fitting anddata presentation were performed using Prism™ software from GraphPadSoftware, Inc.

% phosphorylation data are set forth in FIG. 4. Z′-factor values forvarying kinase concentrations and % phosphorylation of the substrate aredemonstrated in Table 7 below. High Z′-factor values are seen when evenas little as 10% of the substrate is phosphorylated. TABLE 7 Z′-FactorValues for Akt 1 ng Akt 1 kinase per well % Phosphorylation Z′-factorvalue 5.0 8 0.78 10.0 12 0.88 25.0 20 0.91 37.5 28 0.92 42.0 30 0.9250.0 33 0.93 75.0 38 0.93 100.0 43 0.92 150.0 50 0.90b. Abl 1

Abl 1 kinase was serially diluted in a 384 well plate (finalconcentration ranging from 4.9 ng/ml to 10,000 ng/ml). Kinase reactionswere initiated by the addition of a solution of Abl 1 substrate, SEQ IDNO: 3 (modified as described in Example 1b above; 2 μM finalconcentration), and ATP (7 μM final concentration). The plates wereallowed to incubate at room temperature for 1 hour. Chymotrypsin wasthen added to each well and the plates allowed to incubate for 1 hour atroom temperature. The plate was read using a TECAN SAFIRE™monochromator-based fluorescence plate reader (Excitation at 400 nm (12nm bandwidth); Emission at 445 nm (12 nm bandwidth); and Emission at 520nm (12 nm bandwidth). Curve fitting and data presentation were performedusing Prism™ software from GraphPad Software, Inc.

% phosphorylation data are set forth in FIG. 5. Z′-factor values forvarying kinase concentrations and % phosphorylation of the substrate aredemonstrated in Table 8 below. High Z′-factor values are seen when evenas little as 10% of the substrate is phosphorylated. TABLE 8 Z′-FactorValues for Abl 1 Assay ng Abl 1 kinase per well % PhosphorylationZ′-factor value 0.50 10 0.72 0.75 15 0.79 1.00 23 0.84 1.30 30 0.85 1.5035 0.86 2.00 46 0.93 2.50 53 0.94 3.00 61 0.94 3.50 79 0.95c. PKA and PKCα Kinases

Experiments similar to those outlined above were performed for 8compositions of matter (SEQ ID NOs: 47, 50, 43, 58, 59, 60, 63, and 68)against PKA and PKCα FIG. 8 demonstrates the % phosphorylation by theserine/threonine kinase PKA of the 8 compositions against which it wasscreened. As shown, SEQ ID NO: 47 modified as described, is the bestsubstrate for the kinase because it yielded maximal phosphorylation evenat low kinase concentrations.

FIG. 9 demonstrates the % phosphorylation by PKCα of the 8 compositionsagainst which it was screened. As shown, only SEQ ID NO: 60 modified asdescribed, is phosphorylated by this S/T kinase.

Example 3 Identifying Modulators of Kinase Activity

a. Abl 1 (Tyrosine Kinase) Inhibitors

Two test compounds (Staurosporine and Genistein, available fromCalbiochem) were evaluated for their ability to inhibit phosphorylationof an Abl 1 substrate (SEQ ID NO: 3, modified as described in Example 1babove) by Abl 1. Serial dilutions of the respective test compound (finalconcentration ranging from 0.3 to 500,000 nM) were dispensed in 384 wellplates. 5 μL of an Abl 1 kinase/Abl 1 substrate solution (finalconcentration of 1.5 ng Abl 1/well and 2 μM of Abl 1 substrate/well)were added to each well, along with 2.5 μL of ATP (final concentrationof 10 μM). The plate was mixed and incubated for 1 hour at roomtemperature. 5 μL of chymotrypsin were added to each well (finalconcentration of 1 μg/mL), and the plate mixed and allowed to incubateat room temperature for 1 hour. The plate was read using a TECAN SAFIRE™monochromator-based fluorescence plate reader (Excitation at 400 nm (12nm bandwidth); Emission at 445 nm (12 nm bandwidth); and Emission at 520nm (12 nm bandwidth). Curve fitting and data presentation were performedusing Prism™ software from GraphPad Software, Inc.

The dynamic range of the Abl 1 assay was defined by performing 0% and100% inhibition controls. The 0% inhibition control phosphorylated 30%of the Abl 1 substrate, while the 100% inhibition control was a kinasereaction done in the absence of ATP, resulting in nonphosphorylated Abl1 substrate. As a nonphosphorylated Abl1 substrate is cleaved completelyby chymotrypsin, it will exhibit a loss of FRET and a concomitantincrease in the Emissions ratio at 445 nm (coumarin)/520 nm(fluorescein).

FIG. 6 demonstrates the inhibition curves by Staurosporine and Genisteinfor phosphorylation of the Abl 1 substrate by Abl 1. An IC₅₀ value isdefined as the inhibitor concentration that produces a half-maximalshift in the same Emissions ratio. Error bars on FIG. 6 represent onestandard deviation from the mean of three replicates. As can be seenfrom FIG. 6, the IC₅₀ is 33 nM for Staurosporine and 4.1 μM forGenistein.

b. Akt 1 (Serine/Threonine Kinase) Inhibitors

Two test compounds (Staurosporine and Ro-31-8220 (available fromCalbiochem) were evaluated for their ability to inhibit phosphorylationof an Akt 1 substrate (SEQ ID NO: 59, modified as described in Example1a above) by Akt 1. Serial dilutions of the respective test compound(final concentration ranging from 0.019 to 50,000 nM) were dispensed in384 well plates. 5 μL of an Akt 1 kinase/Akt 1 substrate solution (finalconcentration of 42 ng Akt 1/well and 2 μM of Akt 1 substrate/well wereadded to each well, along with 2.5 μL of ATP (final concentration of10.5μM). The plate was mixed and incubated for 1 hour at roomtemperature. 5 μL of chymotrypsin were added to each well (finalconcentration of 100 ng/mL), and the plate mixed and allowed to incubateat room temperature for 1 hour. The plate was read using a TECAN SAFIRE™monochromator-based fluorescence plate reader (Excitation at 400 nm (12nm bandwidth); Emission at 445 nm (12 nm bandwidth); and Emission at 520nm (12 nm bandwidth). Curve fitting and data presentation were performedusing Prism™ software from GraphPad Software, Inc.

The dynamic range of the Akt 1 assay was defined by performing 0% and100% inhibition controls. The 0% inhibition control phosphorylated 30%of the Akt 1 substrate, while the 100% inhibition control was a kinasereaction done in the absence of ATP, resulting in nonphosphorylated Akt1 substrate. As a nonphosphorylated Akt 1 substrate is cleavedcompletely by chymotrypsin, it will exhibit a loss of FRET and aconcomitant increase in the Emissions ratio at 445 nm (coumarin)/520 nm(fluorescein).

FIG. 7 demonstrates the inhibition curves by Staurosporine andRo-31-8220 for phosphorylation of the Akt 1 substrate by Akt 1. An IC₅₀value is defined as the inhibitor concentration that produces ahalf-maximal shift in the same Emissions ratio. Error bars on FIG. 7represent one standard deviation from the mean of three replicates. Ascan be seen from FIG. 7, the IC₅₀ is 17.2 nM for Staurosporine and 408nM for Ro-31-8220.

The experiment was repeated using 15 μM ATP and 3 ng/well Akt 1 and wasalso successful.

Example 4 Panel Assays for Evaluating Substrates for Kinases

Compositions are screened against S/T and Y kinases in a series ofmulti-well (96 or 384 well) plate formats to evaluate their suitabilityas substrates for serine/threonine kinases and/or tyrosine kinases. Eachcomposition has a 7-hydroxycoumarin moiety conjugated to the ε-NH2 ofthe C-terminal lysine and a 5-FAM moiety conjugated to an N-terminalGABA linker.

The following kinases are screened: Akt1, Akt2, Akt3, Aurora A, CaMKII,CDK2/CycA, CDK3/CycE, CDK7/CycH, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1-γ,MSK1, PAK2, PKA, PKG, ROCK, ROCK2, CDK2/CycA, CDK3/CycE, ERK1, ERK2,IKKα, IKKβ, p38β, p38γ, p38δ, REDK, AMPK, CDK6, MAPKAP-K2, MAPKAP-K3,MAPKAP-K5, SGK1, PIM1, CHK1, CHK2, PKCα, PKCβI, PKCβII, PKCγ, PKCδ,PKCε, PKCζ, PKCη, PKCθ, PKCι, and p70 S6 Kinase, Abl1, Abl2, BMX, CSF1R,Csk, EphB4, Fes/Fps, FGFR1, FGFR4, Fgr, FLT3, Fyn, Hck, IGF1R, IRKβ,ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, Src N1, Src N2,SYK, TIE2, TRKa, YES, CK1δ, IKKα, IKKβ, IRTK, CDK1, CDK5/p35, MEK1,MEK5, MEK2, MKK3, MKK4, MKK7, RAF1, GSK3α, GSK3β, MEKK1, CK1δ, CK-1α,CKIIα, JNK1, JNK2, JNK3, and EGFR.

A 384 multiwell plate defines a matrix as follows: each of columns 1-20of the plate correspond to a particular kinase, while rows 1-16 in eachkinase column correspond to duplicate samples of 8 differentcompositions of matter that are potential kinase substrates. Columns21-24 correspond to control columns, representing 0% phosphorylatedcontrols (100% inhibition of kinase, no ATP), and 100% phosphorylatedcontrol (e.g., synthetic phosphorylated composition). For a set of 20kinases, 5 μl of 2× final concentration of each kinase are dispensed toeach row of the respective kinase column of the plate. 5 μL of kinasebuffer are added to control columns 21-24. Each of the compositions ofmatter and ATP are then added to the appropriate rows of each kinasecolumn to result in a final concentration of 2 μM composition/0.8 μM ATPper well; these samples are prepared in duplicate. The compositions ofmatter are also added to the appropriate rows of control columns 23 and24 to result in a final concentration of 2 μM unphosphorylatedcomposition. Phosphorylated compositions of matter are added to theappropriate rows of columns 21 and 22 to result in a final concentrationof 2 μM phosphorylated composition. The plate is then mixed on a shakerand incubated for 1 hour at room temperature to allow kinase reactionsto proceed. 5 μL of chymotrypsin are then added to each well. The plateis mixed on a plate shaker and incubated for 1 hour at room temperature.The plate is read using a TECAN SAFIRE™ monochromator-based fluorescenceplate reader (Excitation at 400 nm (12 nm bandwidth); Emission at 445 nm(12 nm bandwidth); and Emission at 520 nm (12 nm bandwidth). Similarassays with varying kinase concentrations are also performed.

Example 5 Phosphatase Assays

8 compositions of matter were screened against 6 phosphatases toevaluate their suitability as substrates for phosphatases. Thecompositions corresponded to SEQ ID NOs: 31, 97, 100, 103, 109, 110,113, and 118, with each peptide having a 7-hydroxycoumarin moietyconjugated to the ε-NH2 of the C-terminal lysine and a 5-FAM moietyconjugated to an N-terminal GABA linker. The following phosphatases wereused PP1α, PP2A, PP2B, PP2C, PTP1B, and LAR.

Each composition was diluted to 4 μM using the appropriate phosphatasebuffer for each of the phosphatases. Each of the phosphatases wasdiluted by serially titrating (each dilution a 2-fold reduction inphosphatase concentration) using the appropriate phosphatase buffer (asrecommended by the vendor). The volume of each phosphatase dilution was5 μl. 5 μl of each 4 μM composition was added to each serial dilution ofeach phosphatase. The samples were mixed on a plate shaker for 60seconds and incubated at room temperature for one hour. Reactions wereperformed in duplicate. After the one hour phosphatase reaction, 5 μl ofchymotrypsin was added to each reaction. The plate was mixed on a plateshaker and incubated for 1 hour at room temperature. The plate was thenread using a TECAN SAFIRE™ monochromator-based fluorescence plate reader(Excitation at 400 nm (12 nm bandwidth); Emission at 445 nm (12 nmbandwidth); and Emission at 520 nm (12 nm bandwidth) to evaluate whetherany of the compositions were substrates of any of the phosphatases.Appropriate controls for each composition and phosphatase were prepared,corresponding to: the nonphosphorylated version of the composition(untreated); the nonphosphorylated version of the composition treatedwith phosphatase only; the nonphosphorylated version of the compositiontreated with chymotrypsin only; the nonphosphorylated compositiontreated with both phosphatase and chymotrypsin; the phosphorylatedcomposition (untreated); the phosphorylated composition treated withphosphatase alone; and the phosphorylated composition treated withchymotrypsin only.

The results indicated that SEQ ID NOs: 100, 109, and 110 were substratesof PP1α Similarly, SEQ ID NOs: 103, 109, 110, and 113 were substrates ofPP2A; SEQ ID NOs: 100 and 110 were substrates of PP2B; SEQ ID NOs: 100,109, and 110 were substrates of PP2C; and SEQ ID NO: 31 was a substratefor both PTP1B and LAR.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A composition of matter comprising: a) a peptide having a length fromfive to fifty amino acids, said peptide comprising a motif selected fromthe group consisting of: AIYAA, (SEQ ID NO: 1) QDYLS, (SEQ ID NO: 4)EIYGV, (SEQ ID NO: 7) TX₁YVA, where X₁ can be G, (SEQ ID NO: 110) A, orE, EEYIQ, and (SEQ ID NO: 17) DYSQV; and (SEQ ID NO: 20)

b) a first detectable moiety, wherein said first detectable moiety isassociated with said ii peptide.
 2. A composition of matter comprising:a) a peptide having a length from five to fifty amino acids, saidpeptide comprising a motif selected from the group consisting of: (SEQID NO: 145) RR(S/T)L, (SEQ ID NO: 146) L(S/T)TT, (SEQ ID NO: 147)L(S/T)LD, (SEQ ID NO: 148) RX₁(S/T)X₂, where X₁ can be V, A, or Q and X₂can be V or L, (SEQ ID NO: 149) TS(S/T)L, (SEQ ID NO: 150) X₁(S/T)PX₂where X₁ can be P or I and X₂ can be G, K, or D, (SEQ ID NO: 151)(S/T)X₁X₂VA, where X₁ can be A, E, or Q and X₂ can be Y or H, (SEQ IDNO: 152) I(S/T)IAN, (SEQ ID NO: 153) SIA(S/T)I, (SEQ ID NO: 154) (S/T)VPPS*P, where S* is a phosphorylated serine, (SEQ ID NO: 155)DX₁(S/T)X₂, where X₁ can be A or E and X₂ can be I or Q, and (SEQ ID NO:156) D(S/T)QV; and

b) a first detectable moiety, wherein said first detectable moiety isassociated with said peptide.
 3. A composition of matter comprising: a)a peptide having a length from five to fifty amino acids, said peptidecomprising a motif selected from the group consisting of: RRX₁(S/T)L,where X₁ can be F, W, or Y, (SEQ ID NO: 45) LX₁(S/T)TT, where X₁ can beF, W, or Y, (SEQ ID NO: 48) X₁L(S/T)LD, where X₁ can be F, W, or Y, (SEQID NO: 51) RX₁X₂(S/T)X₃, where X₁ can be V, A, or Q, X₂ (SEQ ID NO: 54)can be F, W, or Y, and X₃ can be V or L, TX₁S(S/T)L, where X₁ can be F,W, or Y, (SEQ ID NO: 61) X₁X₂(S/T)PX₃ where X₁ can be P or I, X₂ can(SEQ ID NO: 64) be F, W, or Y, and X₃ can be G, K, or D, X₁(S/T)X₂X₃VA,where X₁ can be F, W, or Y, (SEQ ID NO: 71) X₂ can be A, E, or Q, and X₃can be Y or H, IX₁(S/T)IAN, where X₁ can be F, W, or Y, (SEQ ID NO: 78)SIAX₁(S/T)I, where X₁ can be F, W, or Y, (SEQ ID NO: 81) (S/T) VPPS*P,where S* is a phosphorylated (SEQ ID NO: 84) serine, DX₁X₂(S/T)X₃, whereX₁ can be A or E, X₂ can (SEQ ID NO: 87) be F, W, or Y, and X₃ can be Ior Q, and DX₁(S/T)QV, where X₁ can be F, W, or Y. (SEQ ID NO: 92)


4. The composition of claim 3, further comprising a first detectablemoiety, wherein said first detectable moiety is associated with saidpeptide.
 5. The composition of claim 3, wherein said peptide comprises amotif selected from the group consisting of: (SEQ ID NO: 157) RRF(S/T)L,(SEQ ID NO: 158) LF(S/T)TT, (SEQ ID NO: 159) YL(S/T)LD, (SEQ ID NO: 160)RX₁F(S/T)X₂, where X₁ can be V, A, or Q and X₂ can be V or L, (SEQ IDNO: 161) TFS(S/T)L, (SEQ ID NO: 162) X₁F(S/T)PX₂ where X₁ can be P or Iand X₂ can be G, K, or D, (SEQ ID NO: 163) F(S/T)X₁X₂VA, where X₁ can beA, E, or Q and X₂ can be Y or H, (SEQ ID NO: 164) IF(S/T)IAN, (SEQ IDNO: 165) SIAF(S/T)I, (SEQ ID NO: 166) DX₁F(S/T)X₂, where X₁ can be A orE and X₂ can be I or Q, and (SEQ ID NO: 167) DY(S/T)QV.


6. The composition of claim 1, 2, 4, or 5, wherein said first detectablemoiety is covalently linked to said peptide.
 7. The composition of anyof claims 1-5, wherein said peptide comprises a protease cleavage site.8. The composition of claim 7, wherein said protease cleavage site isselected from the group consisting of a chymotrypsin protease cleavagesite, a caspase 3 protease cleavage site, a cathepsin G proteasecleavage site, a trypsin protease cleavage site, an elastase proteasecleavage site, an endoproteinase Asp-N protease cleavage site, and anendoproteinase Glu-N protease cleavage site.
 9. The composition of claim1, 2, 4, or 5, further comprising a second detectable moiety.
 10. Thecomposition of any of claims 1-5, wherein said peptide has a length from8 to 50 amino acids.
 11. The composition of claim 10, wherein saidpeptide has a length from 8 to 25 amino acids.
 12. The composition ofclaim 11, wherein said peptide has a length from 8 to 15 amino acids.13. The composition of claim 1, 2, 4, or 5, wherein said compositionexhibits an optical property, a magnetic property, or a radioactiveproperty.
 14. The composition of claim 13, wherein said compositionexhibits an optical property selected from the group consisting of amolar extinction coefficient at an excitation wavelength, a quantumefficiency, an excitation spectrum, an emission spectrum, an excitationwavelength maximum, an emission wavelength maximum, a ratio ofexcitation amplitudes at two wavelengths, a ratio of emission amplitudesat two wavelengths, an excited state lifetime, an anisotropy, apolarization of emitted light, a resonance energy transfer, and aquenching of emission at a wavelength.
 15. The composition of claim 14,wherein said optical property is selected from the group consisting of afluorescence excitation spectrum, a fluorescence emission spectrum, afluorescence excitation wavelength maximum, a fluorescence emissionwavelength maximum, a ill ratio of fluorescence excitation amplitudes attwo wavelengths, a ratio of fluorescence emission amplitudes at twowavelengths, a fluorescence excited state lifetime, a fluorescenceanisotropy, and a quenching of fluorescence emission at a wavelength.16. The composition of claim 13, wherein said composition exhibits afluorescence excitation maximum in the range from 250 to 750 nm and afluorescence emission maximum in the range from 450 to 800 nm.
 17. Thecomposition of claim 16, wherein said detectable moiety is selected fromthe group consisting of 5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide,6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate,dichlorotriazinylaminofluorescein,tetramethylrhodamine-5-isothiocyanate,tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein,5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and7-amino-4-methylcoumarin-3-acetic acid.
 18. The composition of claim 1,2, 4, or 5, wherein said first detectable moiety is a member of aspecific binding pair.
 19. The composition of claim 9, wherein saidfirst detectable moiety and said second detectable probe moiety form adark quenching RET pair.
 20. The composition of claim 9, wherein saidfirst detectable moiety and said second detectable moiety form a FRETpair.
 21. The composition of claim 20, wherein said first detectablemoiety is 7-hydroxycoumarin-3-carboxamide and said second detectablemoiety is 5-FAM.
 22. The composition of claim 1, 2, 4, or 5, whereinsaid detectable probe moiety is covalently linked to said peptide via alinker (L).
 23. The composition of claim 19, wherein said L is selectedfrom the group consisting of GABA, diaminopentanyl, and aminohexanoyl.24. The composition of claim 1, wherein said motif is a recognitionmotif for a tyrosine kinase, said recognition motif selected from thegroup consisting of: EAIYAAP, (SEQ ID NO: 2) DQDYLSL, (SEQ ID NO: 5)EEEYIQI, (SEQ ID NO: 18) EEIYGVI, (SEQ ID NO: 8) LTGYVAR, (SEQ ID NO:11) ITAYVAT, (SEQ ID NO: 12) ITEYVAT, (SEQ ID NO: 13) and GDYSQVL. (SEQID NO: 21)


25. The composition of claim 3 or 5, wherein said motif is a recognitionmotif for a serine/threonine kinase, said recognition motif selectedfrom the group consisting of: LRRFSLG, (SEQ ID NO: 46) GLFSTTP, (SEQ IDNO: 49) DYLSLDK, (SEQ ID NO: 52) NRVFSVA, (SEQ ID NO: 55) PRAFSVG, (SEQID NO: 56) RRQFSLR, (SEQ ID NO: 57) RTFSSLA, (SEQ ID NO: 62) APFSPGG,(SEQ ID NO: 65) HPFSPKK, (SEQ ID NO: 66) KIFSPDV, (SEQ ID NO: 67)EFTAYVA, (SEQ ID NO: 72) IFTEYVA, (SEQ ID NO: 73) VFTQHVA, (SEQ ID NO:74) RIFSIANS, (SEQ ID NO: 79) DSIAFSIV, (SEQ ID NO: 82) FSVPPS*PD, whereS* is a (SEQ ID NO: 85) phosphorylated serine, EDAFSII, (SEQ ID NO: 88)EDEFSQN, (SEQ ID NO: 89) and EGDYSQV. (SEQ ID NO: 93)


26. The composition of claim 24, wherein said recognition motif for atyrosine kinase is EAIYAAP (SEQ ID NO: 2), and wherein said tyrosinekinase is selected from the group consisting of Abl1, Abl2, BMX, CSF1R,Csk, EPHB4, Fes/Fps, FGFR1, FGFR4, Fgr, FLT3, Fyn, Hck, IGF1R, IRKβ,ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, Src N1, Src N2,SYK, TIE2, TRKa, and YES.
 27. The composition of claim 24, wherein saidrecognition motif for a tyrosine kinase is DQDYLSL (SEQ ID NO: 5), andwherein said tyrosine kinase is selected from the group consisting ofCaMKII, CDK7/CycH, CK1δ, IKKα, and IKKβ.
 28. The composition of claim25, wherein said recognition motif for a serine/threonine kinase isLRRFSLG (SEQ ID NO: 46), and wherein said serine/threonine kinase isselected from the group consisting of Akt1, Akt2, Akt3, Aurora A,CaMKII, CDK2/CycA, CDK3/CycE, CDK7/CycH, MAPKAP-K1α, MAPKAP-K1β,MAPKAP-K1γ, MSK1, PAK2, PKA, PKQ and ROCK.
 29. The composition of claim25, wherein said recognition motif for a serine/threonine kinase isGLFSTTP (SEQ ID NO: 49), and wherein said serine/threonine kinase isselected from the group consisting of p38γ, p38δ, and REDK.
 30. Thecomposition of claim 25, wherein said recognition motif for aserine/threonine kinase is NRVFSVA (SEQ ID NO: 55), and wherein saidserine/threonine kinase is selected from the group consisting of Akt3,AMPK, CaMKII, CDK7/CycH, CHK2, IKKα, MAPKAP-K1α, MAPKAP-K2, MAPKAP-K3,MAPKAP-K5, PAK2, PKA, PKCβII, REDK, ROCK, ROCK2, and SGK1.
 31. Thecomposition of claim 25, wherein said recognition motif for aserine/threonine kinase is PRAFSVG (SEQ ID NO: 56), and wherein saidserine/threonine kinase is selected from the group consisting of Akt1,Akt2, Akt3, CaMKII, CDK7/CycH, IKKβ, MAPKAP-K1α/RSK1, MAPKAP-K1β/RSK2,MAPKAP-K1γ/RSK3, MSK1, PAK2, PIM1, PKA, PKG, REDK, and SGK1.
 32. Thecomposition of claim 25, wherein said recognition motif for aserine/threonine kinase is RRQFSLR (SEQ ID NO: 57), and wherein saidserine/threonine kinase is selected from the group consisting of Akt1,Akt2, Akt3, CaMKII, CHK1, CHK2, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1γ,MSK1, p70 S6 Kinase, PAK2, PIM1, PKA, PKCα, PKCβI, PKCβI, PKCγ, PKCδ,PKCε, PKCζ, PKCη, PKCθ, PKCι, PKQ ROCK, ROCK2, and SGK1.
 33. Thecomposition of claim 25, wherein said recognition motif for aserine/threonine kinase is RTFSSLA (SEQ ID NO: 62), and wherein saidserine/threonine kinase is selected from the group consisting of Akt1,CDK2/CycA, CDK6, IKKβ, MAPKAP-K1α, MAPKAP-K1β, MAPKAP-K1γ, p70 S6Kinase, PAK2, and PKA.
 34. The composition of claim 25, wherein saidrecognition motif for a serine/threonine kinase is APFSPGG (SEQ ID NO:65), and wherein said serine/threonine kinase is selected from the groupconsisting of CDK2/CycA, CDK3/CycE, ERK1, ERK2, IKKα, p38β, p38γ, andp38δ.
 35. The composition of claim 24, wherein said recognition motiffor a tyrosine kinase is EEIYGVI (SEQ ID NO: 8), and wherein saidtyrosine kinase is selected from the group consisting of Abl1, Abl2,BMX, CSF1R, Csk, EPHB4, Fes/Fps, FGFR1, Fgr, FLT3, Fyn, Hck, IGF1R,IRKβ, IRTK, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, SrcN1, Src N2, SYK, TIE2, TRKa, and YES.
 36. The composition of claim 24,wherein said recognition motif for a tyrosine kinase is LTGYVAR (SEQ IDNO: 11), and wherein said tyrosine kinase is selected from the groupconsisting of CSF1R, FLT3, and c-KIT.
 37. The composition of claim 24,wherein said recognition motif for a tyrosine kinase is EEEYIQI (SEQ IDNO: 18), and wherein said tyrosine kinase is selected from the groupconsisting of EGFR, Zap-70, PDGFR, FGFR4, Abl 1, and Lyn B.
 38. Thecomposition of claim 1, wherein said peptide is selected from the groupconsisting of: EAEAIYAAPGDK, (SEQ ID NO: 3) GDQDYLSLDK, (SEQ ID NO: 6)EEEEYIQIVK, (SEQ ID NO: 19) EEEIYGVIEK, (SEQ ID NO: 9) GVLTGYVARRK, (SEQID NO: 14) DDEITAYVATRK, (SEQ ID NO: 15) TGIITEYVATRK, (SEQ ID NO: 16)and EGDYSQVLEK. (SEQ ID NO: 22)


39. The composition of claim 3, wherein said peptide is selected fromthe group consisting of: ALRRFSLGEK, (SEQ ID NO: 47) VAPFSPGGRAK, (SEQID NO: 68) RGGLFSTTPGGTK, (SEQ ID NO: 50) KLNRVFSVAC, (SEQ ID NO: 58)GDQDYLSLDK, (SEQ ID NO: 53) ARPRAFSVGK, (SEQ ID NO: 59) RRRQFSLRRKAK,(SEQ ID NO: 60) RPRTFSSLAEGK, (SEQ ID NO: 63) AKHPFSPKKAK, (SEQ ID NO:69) DDEFTAYVATRK, (SEQ ID NO: 75) TGIFTEYVATRK, (SEQ ID NO: 76)TGVFTQHVATRK, (SEQ ID NO: 77) QRIFSIANSIVK, (SEQ ID NO: 80)RIDSIAFSIVGK, (SEQ ID NO: 83) PRPFSVPPS*PDK, where S* (SEQ ID NO: 86) isa phosphorylated Serine, EEDAFSIIGK, (SEQ ID NO: 90) REDEFSQNEEK, (SEQID NO: 91) IIKIFSPDVEK, (SEQ ID NO: 70) and EGDYSQVLEK. (SEQ ID NO: 22)


40. The composition of claim 7, wherein said protease cleavage site isFS, FT, or Y.
 41. A method for characterizing a kinase, said methodcomprising: a) contacting a composition of claims 1, 2, or 4 with aprotein kinase under conditions effective for said protein kinase tophosphorylate said composition; and b) measuring the ability of saidprotein kinase to phosphorylate said composition.
 42. A method foridentifying a substrate of a kinase, comprising: a) contacting acomposition of claims 1, 2, or 4 with a protein kinase; b) contactingsaid composition and said protein kinase with a protease to form aprotease mixture; and c) comparing a measurable property in saidprotease mixture with the measurable property in a control proteasemixture lacking said protein kinase, wherein said composition isidentified as a substrate of said protein kinase if said measurableproperty in said protease mixture is different from said measurableproperty in said control protease mixture.
 43. The method of claim 42,wherein ATP is present during said contacting step a).
 44. A method foridentifying a substrate of a kinase, comprising: a) contacting acomposition of claims 1, 2, or 4 with a protein kinase and ATP; b)contacting said composition, said protein kinase, and said ATP with aprotease to form a protease mixture; c) comparing a measurable propertyin said protease mixture with a measurable property in a controlprotease mixture lacking said ATP, wherein said composition isidentified as a substrate of said kinase if said measurable property insaid protease mixture is different from said measurable property in saidcontrol protease mixture.
 45. The method of claim 44, wherein two ormore different compositions of claims 1, 2, or 4 are contactedindependently with said protein kinase and said ATP during saidcontacting step a) to form two or more kinase mixtures, wherein each ofsaid kinase mixtures is contacted independently with a protease duringsaid contacting step b) to form two or more protease mixtures, andwherein a measurable property in each of said protease mixtures iscompared with the measurable property in a corresponding controlmixture.
 46. The method of claim 44, wherein two or more differentprotein kinases are contacted independently with said composition andsaid ATP during said contacting step a) to form two or more kinasemixtures, wherein each of said kinase mixtures is contactedindependently with a protease during said contacting step b) to form twoor more protease mixtures, and wherein a measurable property in each ofsaid protease mixtures is compared with the measurable property in acorresponding control mixture.
 47. The method of claim 44, wherein saidcomparing step c) occurs concurrently with said contacting step b). 48.The method of claim 44, wherein said comparing step c) occurs after saidcontacting step b).
 49. The method of claim 48, wherein said contactingstep b) is completed by inhibiting a proteolytic activity of saidprotease.
 50. The method of claim 49, wherein said proteolytic activityis inhibited by adding a reagent to said protease mixtures or by heatingsaid protease mixtures.
 51. The method of claim 50, wherein said reagentis aprotinin, PMSF, TPCK, AEBSF, chymotrypsin inhibitor 1, andchymotrypsin inhibitor
 2. 52. A method for identifying a modulator ofactivity of a kinase, comprising: a) forming a mixture of a proteinkinase, a substrate for said protein kinase, and a test compound; b)contacting said mixture with a protease to form a protease mixture; andc) comparing a measurable property in said protease mixture to themeasurable property in a control mixture of said substrate, said proteinkinase, and said protease in the absence of said test compound, whereinsaid test compound is identified as a modulator of activity of saidkinase if said measurable property in said protease mixture is differentfrom said measurable property in said control mixture.
 53. The method ofclaim 52, wherein ATP is present during said contacting step a).
 54. Amethod for identifying a modulator of activity of a kinase, comprising:a) contacting a protein kinase, a substrate for said protein kinase,ATP, and a test compound to form a kinase mixture; b) contacting saidkinase mixture with a protease to form a protease mixture; c) comparinga measurable property in said protease mixture with the measurableproperty in a control mixture comprising said composition, said proteinkinase, and said protease in the absence of said test compound, whereinsaid composition is identified as a modulator of activity of said kinaseif said measurable property in said protease mixture is different fromsaid measurable property in said control mixture.
 55. The method ofclaim 54, wherein two or more different test compounds are contactedindependently with said protein kinase, said ATP, and said substrate insaid contacting step a) to form two or more kinase mixtures, whereineach of said kinase mixtures is contacted independently with a proteasein said contacting step b) to form two or more protease mixtures, andwherein a measurable property in each of said protease mixtures iscompared with the measurable property in a corresponding controlmixture.
 56. The method of claim 54, wherein two or more differentprotein kinases are contacted independently with said ATP, said testcompound, and said substrate in said contacting step a) to form two ormore kinase mixtures, wherein each of said kinase mixtures is contactedindependently with a protease in said contacting step b) to form two ormore protease mixtures, and wherein a measurable property in each ofsaid protease mixtures is compared with the measurable property in acorresponding control mixture.
 57. The method of claim 54, wherein saidcomparing step c) occurs during said contacting step b).
 58. The methodof claim 54, wherein said comparing step c) occurs after said contactingstep b).
 59. The method of claim 58, wherein said contacting step b) iscompleted by inhibiting a proteolytic activity of said protease.
 60. Themethod of claim 59, wherein said proteolytic activity is inhibited byadding a reagent to said protease mixtures or by heating said proteasemixtures.
 61. The method of claim 60, wherein said reagent is aprotinin,PMSF, TPCK, AEBSF, chymotrypsin inhibitor 1, and chymotrypsin inhibitor2.
 62. The composition of claim 1, wherein said Y in said motif isphosphorylated.
 63. The composition of claim 2, 3, 4, or 5, wherein saidS/T in said motif is phosphorylated.
 64. The composition of claim 24,wherein said recognition motif for a tyrosine kinase is phosphorylatedat the Y, and wherein said phosphorylated recognition motif is arecognition motif for a protein phosphatase.
 65. The composition ofclaim 5, wherein said peptide comprises a protein phosphataserecognition motif selected from the group consisting of: LRRFS*LG, (SEQID NO: 96) GLFS*TTP, (SEQ ID NO: 99) DYLS*LDK, (SEQ ID NO: 102)NRVFS*VA, (SEQ ID NO: 105) PRAFS*VG, (SEQ ID NO: 106) RRQFS*LR, (SEQ IDNO: 107) RTFSS*LA, (SEQ ID NO: 112) APFS*PGG, (SEQ ID NO: 115) HPFS*PKK,(SEQ ID NO: 116) KIFS*PDV, (SEQ ID NO: 117) EFT*AYVA, (SEQ ID NO: 122)IFT*EYVA, (SEQ ID NO: 123) VFT*QHVA, (SEQ ID NO: 124) RIFS*IANS (SEQ IDNO: 129) DSIAFS*IV, (SEQ ID NO: 132) FS*VPPS*PD, (SEQ ID NO: 135)EDAFS*II, (SEQ ID NO: 138) EDEFS*QN, (SEQ ID NO: 139) and EGDYS*QV, (SEQID NO: 143)

wherein S* represents a phosphorylated serine and T* represents aphosphorylated threonine.
 66. The composition of claim 1, wherein saidpeptide comprises a protein phosphatase recognition motif, and whereinsaid peptide is selected from the group consisting of: EAEAIY*AAPGDK,(SEQ ID NO: 25) GDQDY*LSLDK, (SEQ ID NO: 28) EEEEY*IQIVK, (SEQ ID NO:41) EEEIY*GVIEK, (SEQ ID NO: 31) GVLTGY*VARRK, (SEQ ID NO: 36)DDEITAY*VATRK, (SEQ ID NO: 37) TGIITEY*VATRK, (SEQ ID NO: 38) andEGDY*SQVLEK, (SEQ ID NO: 44)

wherein Y* represents a phosphorylated tyrosine.
 67. The composition ofclaim 3, wherein said peptide has a sequence selected from the groupconsisting of: ALRRFS*LGEK, (SEQ ID NO: 97) VAPFS*PGGRAK, (SEQ ID NO:118) RGGLFS*TTPGGTK, (SEQ ID NO: 100) KLNRVFS*VAC, (SEQ ID NO: 108)GDQDYLS*LDK, (SEQ ID NO: 103) ARPRAFS*VGK, (SEQ ID NO: 109)RRRQFS*LRRKAK, (SEQ ID NO: 110) RPRTFSS*LAEGK, (SEQ ID NO: 113)AKHPFS*PKKAK, (SEQ ID NO: 119) DDEFT*AYVATRK, (SEQ ID NO: 125)TGIFT*EYVATRK, (SEQ ID NO: 126) TGVFT*QHVATRK, (SEQ ID NO: 127)QRIFS*IANSIVK, (SEQ ID NO: 130) RIDSIAFS*IVGK, (SEQ ID NO: 133)PRPFS*VPPS*PDK, (SEQ ID NO: 136) EEDAFS*IIGK, (SEQ ID NO: 140)REDEFS*QNEEK, (SEQ ID NO: 141) IIKIFS*PDVEK, (SEQ ID NO: 120) andEGDYS*QVLEK, (SEQ ID NO: 144)

wherein S* represents a phosphorylated serine and T* represents aphosphorylated threonine.
 68. The composition of claim 64, wherein saidphosphatase recognition motif is EAIY*AAP, wherein Y* is aphosphorylated tyrosine (SEQ ID NO: 24), and wherein said phosphatase isselected from the group consisting of PTP1B, LAR, and LCA.
 69. Thecomposition of claim 65, wherein said phosphatase recognition motif isDQDYLS*L, wherein S* is a phosphorylated serine (SEQ ID NO: 27), andwherein said phosphatase is selected from the group consisting of PP1α,PP2A, PP2B, and PP2C.
 70. The composition of claim 65, wherein saidphosphatase recognition motif is LRRFS*LG, wherein S* is aphosphorylated serine (SEQ ID NO: 96), and wherein said phosphatase isselected from the group consisting of PP1α, PP2A, and PP2C.
 71. Thecomposition of claim 65, wherein said phosphatase recognition motif isGLFS*TTP, wherein S* is a phosphorylated serine (SEQ ID NO: 99), andwherein said phosphatase is selected from the group consisting of PP1α,PP2A, PP2B, and PP2C.
 72. The composition of claim 65, wherein saidphosphatase recognition motif is NRVFS*VA, wherein S* is aphosphorylated serine (SEQ ID NO: 105), and wherein said phosphatase isselected from the group consisting of PP1α, PP2A, PP2B, and PP2C. 73.The composition of claim 65, wherein said phosphatase recognition motifis PRAFS*VC; wherein S* is a phosphorylated serine (SEQ ID NO: 106), andwherein said phosphatase is selected from the group consisting of PP1α,PP2A, and PP2B.
 74. The composition of claim 65, wherein saidphosphatase recognition motif is RRQFS*LR, wherein S* is aphosphorylated serine (SEQ ID NO: 107), and wherein said phosphatase isselected from the group consisting of PP1α, PP2A, and PP2B.
 75. Thecomposition of claim 65, wherein said phosphatase recognition motif isRTFSS*LA, wherein S* is a phosphorylated serine (SEQ ID NO: 112), andwherein said phosphatase is selected from the group consisting of PP1α,PP2A, and PP2B.
 76. The composition of claim 65, wherein saidphosphatase recognition motif is APFS*PGQ wherein S* is a phosphorylatedserine (SEQ ID NO: 115), and wherein said phosphatase is selected fromthe group consisting of PP1α and PP2A.
 77. The composition of claim 64,wherein said phosphatase recognition motif is EEIY*GVI, wherein Y* is aphosphorylated tyrosine (SEQ ID NO: 30), and wherein said phosphatase isselected from the group consisting of PTP1B, LAR, and LCA.
 78. Thecomposition of claim 64, wherein said phosphatase recognition motif isLTGY*VAR, wherein Y* is a phosphorylated tyrosine (SEQ ID NO: 33), andwherein said phosphatase is selected from the group consisting of PTP1B,LAR, and LCA.
 79. A method for characterizing a phosphatase, said methodcomprising: a) contacting a composition of claim 62 with a proteinphosphatase under conditions effective for said protein phosphatase todephosphorylate said composition; and b) measuring the ability of saidprotein phosphatase to dephosphorylate said composition.
 80. A methodfor identifying a substrate of a phosphatase, comprising: a) contactinga composition of claim 62 with a protein phosphatase; b) contacting saidcomposition and said protein phosphatase with a protease to form aprotease mixture; and c) comparing a measurable property in saidprotease mixture with a measurable property in a control proteasemixture lacking said phosphatase, wherein said composition is identifiedas a substrate of said phosphatase if said measurable property in saidprotease mixture is different from said measurable property in saidcontrol protease mixture.
 81. The method of claim 80, wherein two ormore different compositions of claim 62 or claim 63 are contactedindependently with said phosphatase during said contacting step a) toform two or more phosphatase mixtures, wherein each of said phosphatasemixtures is contacted independently with a protease during saidcontacting step b) to form two or more protease mixtures, and wherein ameasurable property in each of said protease mixtures is compared withthe measurable property in a corresponding control mixture.
 82. Themethod of claim 80, wherein two or more different phosphatases arecontacted independently with said composition during said contactingstep a), wherein each of said phosphatase mixtures is contactedindependently with a protease during said contacting step b) to form twoor more protease mixtures, and wherein a measurable property in each ofsaid protease mixtures is compared with the measurable property in acorresponding control mixture.
 83. A method for identifying a modulatorof activity of a phosphatase, comprising: a) contacting a mixture of aprotein phosphatase, a substrate for said protein phosphatase, and atest compound to form a phosphatase mixture; b) contacting saidphosphatase mixture with a protease to form a protease mixture; and c)comparing a measurable property in said protease mixture to themeasurable property in a control protease mixture lacking said testcompound, wherein said test compound is identified as a modulator ofactivity of said phosphatase if said measurable property in saidprotease mixture is different from said measurable property in saidcontrol mixture.
 84. The method of claim 83, wherein two or moredifferent test compounds are contacted independently with saidphosphatase and said substrate during said contacting step a) to formtwo or more phosphatase mixtures, wherein each of said phosphatasemixtures is contacted independently with a protease during saidcontacting step b) to form two or more protease mixtures, and wherein ameasurable property in each of said protease mixtures is compared withthe measurable property in a corresponding control mixture.
 85. Themethod of claim 83, wherein two or more different phosphatases arecontacted independently with said test compound and said substrateduring said contacting step a) to form two or more phosphatase mixtures,wherein each of said phosphatase mixtures is contacted independentlywith a protease during said contacting step b) to form two or moreprotease mixtures, and wherein a measurable property in each of saidprotease mixtures is compared with the measurable property in acorresponding control mixture.
 86. An article of manufacture comprising:a) packaging matter; and b) a composition of matter according to claim1, 2, 4, 5 or 62 associated with said packaging material.
 87. Thearticle of claim 86, further comprising a protein kinase or a proteinphosphatase.
 88. The article of claim 86, further comprising a protease.89. The article of claim 87, further comprising ATP.
 90. The compositionof claim 18, wherein said member of a specific binding pair is anepitope.
 91. The composition of claim 18, wherein said member of aspecific binding pair is biotin.